EP4326856A1 - Stem cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereof - Google Patents
Stem cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereofInfo
- Publication number
- EP4326856A1 EP4326856A1 EP22790648.4A EP22790648A EP4326856A1 EP 4326856 A1 EP4326856 A1 EP 4326856A1 EP 22790648 A EP22790648 A EP 22790648A EP 4326856 A1 EP4326856 A1 EP 4326856A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- cell
- stem
- progenitor
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 361
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 174
- 108091008874 T cell receptors Proteins 0.000 title claims abstract description 105
- 238000000034 method Methods 0.000 title claims abstract description 104
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 title claims abstract description 91
- 210000004027 cell Anatomy 0.000 claims abstract description 335
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 148
- 239000000427 antigen Substances 0.000 claims abstract description 81
- 108091007433 antigens Proteins 0.000 claims abstract description 75
- 102000036639 antigens Human genes 0.000 claims abstract description 75
- 210000003719 b-lymphocyte Anatomy 0.000 claims abstract description 70
- 108091008875 B cell receptors Proteins 0.000 claims abstract description 61
- 230000008707 rearrangement Effects 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims description 101
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 88
- 102000004169 proteins and genes Human genes 0.000 claims description 62
- 101001061851 Homo sapiens V(D)J recombination-activating protein 2 Proteins 0.000 claims description 56
- 230000006798 recombination Effects 0.000 claims description 55
- 238000005215 recombination Methods 0.000 claims description 55
- 150000007523 nucleic acids Chemical class 0.000 claims description 49
- 102100029591 V(D)J recombination-activating protein 2 Human genes 0.000 claims description 48
- 108020004707 nucleic acids Proteins 0.000 claims description 47
- 102000039446 nucleic acids Human genes 0.000 claims description 47
- 102000001183 RAG-1 Human genes 0.000 claims description 46
- 108060006897 RAG1 Proteins 0.000 claims description 42
- 230000002829 reductive effect Effects 0.000 claims description 34
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 31
- 201000010099 disease Diseases 0.000 claims description 30
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 29
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 29
- 102100028156 Non-homologous end-joining factor 1 Human genes 0.000 claims description 29
- 206010028980 Neoplasm Diseases 0.000 claims description 28
- 101000578059 Homo sapiens Non-homologous end-joining factor 1 Proteins 0.000 claims description 26
- 238000012258 culturing Methods 0.000 claims description 26
- 210000001778 pluripotent stem cell Anatomy 0.000 claims description 25
- 102100027828 DNA repair protein XRCC4 Human genes 0.000 claims description 24
- 101710147736 DNA repair protein XRCC4 Proteins 0.000 claims description 24
- 102000005768 DNA-Activated Protein Kinase Human genes 0.000 claims description 24
- 108010006124 DNA-Activated Protein Kinase Proteins 0.000 claims description 24
- 102100036663 Protein PAXX Human genes 0.000 claims description 24
- 101710168726 Protein PAXX Proteins 0.000 claims description 24
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 23
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 23
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 21
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 21
- 210000001978 pro-t lymphocyte Anatomy 0.000 claims description 13
- 230000003612 virological effect Effects 0.000 claims description 13
- 108010060248 DNA Ligase ATP Proteins 0.000 claims description 12
- 210000005260 human cell Anatomy 0.000 claims description 9
- 201000011510 cancer Diseases 0.000 claims description 8
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 6
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 6
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 5
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 5
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 5
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 5
- 102000008158 DNA Ligase ATP Human genes 0.000 claims 5
- 239000000203 mixture Substances 0.000 abstract description 14
- 101150049556 Bcr gene Proteins 0.000 abstract description 4
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 57
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 57
- 102100026964 M1-specific T cell receptor beta chain Human genes 0.000 description 56
- 108010087408 alpha-beta T-Cell Antigen Receptors Proteins 0.000 description 51
- 210000003317 double-positive, alpha-beta immature T lymphocyte Anatomy 0.000 description 31
- 239000000523 sample Substances 0.000 description 19
- 230000004069 differentiation Effects 0.000 description 18
- 230000004083 survival effect Effects 0.000 description 18
- 102000004127 Cytokines Human genes 0.000 description 17
- 108090000695 Cytokines Proteins 0.000 description 17
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 16
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 15
- 238000012217 deletion Methods 0.000 description 13
- 230000037430 deletion Effects 0.000 description 12
- 101150075516 RAG2 gene Proteins 0.000 description 11
- 230000001105 regulatory effect Effects 0.000 description 11
- 239000005090 green fluorescent protein Substances 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 9
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 230000002950 deficient Effects 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 239000002243 precursor Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 102100033195 DNA ligase 4 Human genes 0.000 description 7
- 108010002586 Interleukin-7 Proteins 0.000 description 7
- 102000000704 Interleukin-7 Human genes 0.000 description 7
- 239000004698 Polyethylene Substances 0.000 description 7
- 206010043276 Teratoma Diseases 0.000 description 7
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 7
- 210000004700 fetal blood Anatomy 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 229940100994 interleukin-7 Drugs 0.000 description 7
- 101150013400 rag1 gene Proteins 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 108700028369 Alleles Proteins 0.000 description 6
- 238000003559 RNA-seq method Methods 0.000 description 6
- 238000003501 co-culture Methods 0.000 description 6
- 239000003636 conditioned culture medium Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 108700014844 flt3 ligand Proteins 0.000 description 6
- 102000048797 human RAG2 Human genes 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108091033409 CRISPR Proteins 0.000 description 5
- 108020005004 Guide RNA Proteins 0.000 description 5
- 230000008236 biological pathway Effects 0.000 description 5
- 230000024245 cell differentiation Effects 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 210000002242 embryoid body Anatomy 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108010032099 V(D)J recombination activating protein 2 Proteins 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000011712 cell development Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 210000001541 thymus gland Anatomy 0.000 description 4
- 230000009258 tissue cross reactivity Effects 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 3
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 3
- 101710127639 Non-homologous end-joining factor 1 Proteins 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000003511 endothelial effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000003394 haemopoietic effect Effects 0.000 description 3
- 210000003566 hemangioblast Anatomy 0.000 description 3
- 230000006780 non-homologous end joining Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- 239000012117 Alexa Fluor 700 Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 101150041972 CDKN2A gene Proteins 0.000 description 2
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 2
- 241000718430 Comocladia glabra Species 0.000 description 2
- -1 Cpf1 Proteins 0.000 description 2
- 101150097493 D gene Proteins 0.000 description 2
- 102100027641 DNA-binding protein inhibitor ID-1 Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 102100038614 Hemoglobin subunit gamma-1 Human genes 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101001081590 Homo sapiens DNA-binding protein inhibitor ID-1 Proteins 0.000 description 2
- 101001031977 Homo sapiens Hemoglobin subunit gamma-1 Proteins 0.000 description 2
- 101000998139 Homo sapiens Interleukin-32 Proteins 0.000 description 2
- 101001043807 Homo sapiens Interleukin-7 Proteins 0.000 description 2
- 101000973618 Homo sapiens NF-kappa-B essential modulator Proteins 0.000 description 2
- 101000686034 Homo sapiens Nuclear receptor ROR-gamma Proteins 0.000 description 2
- 101000666661 Homo sapiens Rho-related GTP-binding protein RhoU Proteins 0.000 description 2
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 2
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 2
- 102100033501 Interleukin-32 Human genes 0.000 description 2
- 101150008942 J gene Proteins 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 108050008953 Melanoma-associated antigen Proteins 0.000 description 2
- 102100022219 NF-kappa-B essential modulator Human genes 0.000 description 2
- 102100023421 Nuclear receptor ROR-gamma Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 102100038399 Rho-related GTP-binding protein RhoU Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 2
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 2
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 2
- 101150117115 V gene Proteins 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 102100026497 Zinc finger protein 654 Human genes 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000002306 biochemical method Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000000925 erythroid effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 230000037433 frameshift Effects 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 210000001654 germ layer Anatomy 0.000 description 2
- 102000052622 human IL7 Human genes 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- SIVJKYRAPQKLIM-UHFFFAOYSA-N 3-(3,4-difluorophenyl)-n-(3-fluoro-5-morpholin-4-ylphenyl)propanamide Chemical compound C=1C(N2CCOCC2)=CC(F)=CC=1NC(=O)CCC1=CC=C(F)C(F)=C1 SIVJKYRAPQKLIM-UHFFFAOYSA-N 0.000 description 1
- 102100032292 A disintegrin and metalloproteinase with thrombospondin motifs 17 Human genes 0.000 description 1
- 108091005674 ADAMTS17 Proteins 0.000 description 1
- 102100039181 Ankyrin repeat domain-containing protein 1 Human genes 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 102000008682 Argonaute Proteins Human genes 0.000 description 1
- 108010088141 Argonaute Proteins Proteins 0.000 description 1
- 102100021933 C-C motif chemokine 25 Human genes 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102100039553 CKLF-like MARVEL transmembrane domain-containing protein 8 Human genes 0.000 description 1
- 102100030621 Carboxypeptidase A4 Human genes 0.000 description 1
- 102100026658 Cathepsin W Human genes 0.000 description 1
- 229940123587 Cell cycle inhibitor Drugs 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102100025816 Coiled-coil domain-containing protein 152 Human genes 0.000 description 1
- 102100031043 Coiled-coil domain-containing protein 8 Human genes 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 210000001086 DN3 alpha-beta immature T lymphocyte Anatomy 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 101150078651 Epha4 gene Proteins 0.000 description 1
- 102100021616 Ephrin type-A receptor 4 Human genes 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 102100021245 G-protein coupled receptor 183 Human genes 0.000 description 1
- 101001077417 Gallus gallus Potassium voltage-gated channel subfamily H member 6 Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102100038617 Hemoglobin subunit gamma-2 Human genes 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101000889396 Homo sapiens Ankyrin repeat domain-containing protein 1 Proteins 0.000 description 1
- 101000897486 Homo sapiens C-C motif chemokine 25 Proteins 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000888512 Homo sapiens CKLF-like MARVEL transmembrane domain-containing protein 8 Proteins 0.000 description 1
- 101000772572 Homo sapiens Carboxypeptidase A4 Proteins 0.000 description 1
- 101000910988 Homo sapiens Cathepsin W Proteins 0.000 description 1
- 101000932658 Homo sapiens Coiled-coil domain-containing protein 152 Proteins 0.000 description 1
- 101000777367 Homo sapiens Coiled-coil domain-containing protein 8 Proteins 0.000 description 1
- 101001040801 Homo sapiens G-protein coupled receptor 183 Proteins 0.000 description 1
- 101001031961 Homo sapiens Hemoglobin subunit gamma-2 Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101001033312 Homo sapiens Interleukin-4 receptor subunit alpha Proteins 0.000 description 1
- 101001067187 Homo sapiens Plexin-A2 Proteins 0.000 description 1
- 101001105486 Homo sapiens Proteasome subunit alpha type-7 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 101001010792 Homo sapiens Transcriptional regulator ERG Proteins 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 1
- 108700003107 Interleukin-1 Receptor-Like 1 Proteins 0.000 description 1
- 102100036706 Interleukin-1 receptor-like 1 Human genes 0.000 description 1
- 108010017411 Interleukin-21 Receptors Proteins 0.000 description 1
- 102100030699 Interleukin-21 receptor Human genes 0.000 description 1
- 102100039078 Interleukin-4 receptor subunit alpha Human genes 0.000 description 1
- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100034256 Mucin-1 Human genes 0.000 description 1
- 108010008707 Mucin-1 Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 101100058550 Mus musculus Bmi1 gene Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102100034381 Plexin-A2 Human genes 0.000 description 1
- 102100022807 Potassium voltage-gated channel subfamily H member 2 Human genes 0.000 description 1
- 208000009052 Precursor T-Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 208000017414 Precursor T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102100021201 Proteasome subunit alpha type-7 Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 108700042075 T-Cell Receptor Genes Proteins 0.000 description 1
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 101150002618 TCRP gene Proteins 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 102100032120 Toll/interleukin-1 receptor domain-containing adapter protein Human genes 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102100039094 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 101710160666 Vascular cell adhesion protein 1 Proteins 0.000 description 1
- 101150097457 Vcam1 gene Proteins 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 101150007302 dntt gene Proteins 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 210000002861 immature t-cell Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 108010065059 methylaspartate ammonia-lyase Proteins 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000024639 negative regulation of intrinsic apoptotic signaling pathway Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000030454 positive regulation of lymphocyte activation Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006716 regulation of lymphocyte proliferation Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2307—Interleukin-7 (IL-7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Definitions
- the application relates to stem cells that are unable to undergo T cell receptor (TCR) or B cell receptor (BCR) gene rearrangements.
- TCR T cell receptor
- BCR B cell receptor
- the application relates to methods, compositions and kits for use in generating cells of the T cell lineage or B cell lineage comprising an unrearranged TCR gene locus or BCR gene locus, respectively.
- T cells to specifically recognize antigens is accomplished through the expression of specific T cell receptors encoded by the uniquely rearranged genomic loci of the TCRa and b chains. T cell development is predicated on the successful rearrangement of the T cell receptor (TCR) gene loci, which encode for antigen-specific receptors.
- TCR T cell receptor
- V(D)J Recombination Activating Genes (RAG) 1 and 2 are two essential DNA processing enzymes required for the rearrangement of the B cell receptor and T cell receptor gene loci (Schatz at al., 1989; Oettinger et al, 1990).
- RAG 1/2 initiate V(D)J recombination by forming a complex that first recognizes and binds to recombination signal sequences (RSS) found adjacent to each V, D, and J gene segment.
- RSS recombination signal sequences
- the RAG complex Upon formation of a synapse with another RSS, the RAG complex induces a double-strand DNA break, which is repaired by non-homologous end-joining process (Jones and Gellert, 2004; Smith et al, 2019). This ultimately results in the imperfect joining of different V, D, and J gene segments to potentially generate millions of different antigen receptors from a few hundred V(D)J segments.
- the inventors differentiated human pluripotent stem cells with a
- RAG2-KO CRISPR/Cas9-directed deletion of the RAG2 gene
- the disclosure provides a method of generating stem or progenitor cells unable to undergo T cell receptor (TCR) gene rearrangements (TCR), the method comprising:
- the method further comprises (b) isolating cells of the T cell lineage.
- V(D)J recombination is RAG1 and/or RAG2.
- the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- PAXXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- the stem cells are pluripotent stem cells.
- the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPSCs).
- the stem cells or progenitor cells are human cells.
- the cells of the T cell lineage are progenitor T (proT) cells, optionally CD45+CD34+CD7+ progenitor T (proT) cells.
- the cells of the T cell lineage are CD4+CD8+ double positive cells or CD4+CD8+CD3+ double positive cells.
- the cells of the T cell lineage are CD8+CD3+ single positive cells or CD4+CD3+ single positive cells.
- the method further comprises engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise at least one of a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
- TCR T cell receptor
- CAR chimeric antigen receptor
- the stem cells or progenitor cells or the cells of the T cell lineage express the at least one of a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
- TCR T cell receptor
- CAR chimeric antigen receptor
- the method further comprises engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise a nucleic acid encoding a TCRp chain.
- the stem cells or progenitor cells or the cells of the T cell lineage that comprise a nucleic acid encoding a TCRp chain do not comprise a nucleic acid that encodes a TCRp chain or a chimeric antigen receptor (CAR).
- the stem cells or progenitor cells or the cells of the T cell lineage express a TCRp chain.
- the stem cells or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp chain and a nucleic acid encoding a CAR.
- the TCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
- the disclosure also provides a cell of the T cell lineage, wherein the cell is generated by a method described herein.
- the cell of the T cell lineage is a CD4+CD8+ double positive cell or a CD4+CD8+CD3+ double positive cell.
- the cell is a CD45+CD34+CD7+ progenitor T cell, CD8+CD3+ single positive cell or CD4+CD3+ single positive cell.
- the disclosure also provides a stem or progenitor cell, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cell or progenitor cell is reduced or eliminated compared to a wild-type stem cell or progenitor cell.
- the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
- the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for
- V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- PAXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- the stem or progenitor cell further comprises at least one of a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
- TCR T cell receptor
- CAR chimeric antigen receptor
- the stem or progenitor cell further comprises a nucleic acid encoding a TCRp chain.
- the stem or progenitor cell that comprises a nucleic acid encoding a TCRp chain does not comprise a nucleic acid that encodes a TCRp chain or a chimeric antigen receptor (CAR).
- the stem cell is a pluripotent stem cell, optionally an embryonic stem cell or induced pluripotent stem cell (iPSC).
- iPSC induced pluripotent stem cell
- the stem cell or progenitor cell is a human cell.
- the disclosure also provides a use of a stem or progenitor cell as described herein for generating cells of the T cell lineage.
- the disclosure also provides a kit comprising (i) a stem or progenitor cell as described herein and (ii) instructions for use of the stem or progenitor cell as described herein for generating cells of the T cell lineage.
- the disclosure further provides a method of treating a disease or condition in a subject comprising:
- stem cells or progenitor cells are engineered to comprise at least one of a nucleic acid encoding a T cell receptor (TCR) and a chimeric antigen receptor (CAR) that confers specificity to an antigen.
- TCR T cell receptor
- CAR chimeric antigen receptor
- the disclosure also provides a method of treating a disease or condition in a subject comprising: (i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the T cell lineage wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
- TCR T cell receptor
- CAR chimeric antigen receptor
- the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
- the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for the at least one gene or protein required for
- V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- PAXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- the disease is cancer and the antigen is a tumor- associated antigen.
- the disclosure also provides a method of generating stem or progenitor cells unable to undergo T cell receptor (BCR) gene rearrangements, the method comprising:
- the method further comprises (b) isolating cells of the B cell lineage.
- V(D)J recombination is RAG1 and/or RAG2.
- V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- PAXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- the stem cells are pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells (iPSCs).
- the stem cells or progenitor cells are human cells.
- the cells of the B cell lineage are CD20+ or
- the cells of the B cell lineage are Tumor-induced
- TIBs Infiltrating B Cells
- the method further comprises engineering the stem cells or progenitor cells or the cells of the B cell lineage to comprise at least one of a nucleic acid encoding a B cell receptor (BCR), a BCRp chain and a chimeric antigen receptor (CAR).
- BCR B cell receptor
- CAR chimeric antigen receptor
- the stem cells or progenitor cells or the cells of the B cell lineage express the B cell receptor (BCR), the BCRp chain or the chimeric antigen receptor (CAR).
- BCR B cell receptor
- CAR chimeric antigen receptor
- the BCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
- the disclosure also provides a cell of the B cell lineage, wherein the cell is generated by a method as described herein.
- the cell of the B cell lineage is a CD20+ or CD19+ cell.
- the cell of the B cell lineage is a Tumor-Infiltrating
- TIB B Cell
- the disclosure also provides a use of a stem or progenitor cell as described herein for generating cells of the B cell lineage.
- the disclosure also provides a kit comprising (i) a stem or progenitor cell as described herein and (ii) instructions for use of the stem or progenitor cell as described herein for generating cells of the B cell lineage. [0054] The disclosure also provides a method of treating a disease or condition in a subject comprising:
- stem cells or progenitor cells are engineered to comprise a nucleic acid encoding a B cell receptor (BCR) that confers specificity to an antigen.
- BCR B cell receptor
- the disclosure also provides a method of treating a disease or condition in a subject comprising:
- the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
- V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- PAXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- the disease is cancer and the antigen is a tumor- associated antigen.
- the antigen is a tumor-associated antigen.
- Fig. 1 shows generation and characterization of RAG2-KO hESC lines.
- Fig. 1A shows Sanger Sequencing of genomic DNA of CRISPR/Cas9- targeted RAG2 of clones 1 and 4. The altered allele is depicted in chromatograph and sequence above it, while alignment of the altered and WT alleles are shown below.
- Fig. 1B shows an immunoblot for RAG2 protein expression of in v/fro-derived T-lineage cells from RAG2-KO clones (1 and 4) and Control WT hESCs, or Control PBMCs obtained from a T- ALL patient. GAPDH serves as a loading control.
- Fig. 1A shows Sanger Sequencing of genomic DNA of CRISPR/Cas9- targeted RAG2 of clones 1 and 4. The altered allele is depicted in chromatograph and sequence above it, while alignment of the altered and WT alleles are shown below.
- Fig. 1B shows an immunoblot for RAG2 protein expression of in
- FIG. 1C shows immunofluorescence of RAG2-KO hESC clones for pluripotency markers. The scale bar corresponds to 100 pm.
- Fig. 1 D shows immunohistochemistry of RAG2-KO hESC clones against ectoderm (neural tissue), mesoderm (cartilage), and endoderm (glandular tissue) lineages of a teratoma formation assay.
- Fig. 2 (A-B) shows T cell development from Control WT and RAG2-
- Fig. 3 shows forced expression of a rearranged TCRp chain in
- FIG. 3A shows a schematic of the experimental approach.
- Fig. 3B shows cell counts of RAG2-KO DPs (clones 1 and 4) retrovirally-transduced with empty vector (dTomato), TCR alpha chain (TRA 1383i), or TCR beta chain (TRB 1383i) per 10,000 input of CD34 + cells.
- Fig. 4 shows TCR gene expression and CD4 + CD8 + signature gene expression.
- Fig. 5A shows a heatmap analysis of specific TCR genes expressed in Control WT DPs but absent in RAG2-K01/4 DPs and TCRp-transduced RAG2-K01/4 DPs.
- Fig. 5 shows transcriptomic analysis of Control WT, RAG2-
- Fig. 6 (A-B) shows an analysis of proliferation and differentiation genes and biological pathways.
- Fig. 6A is a heatmap analysis of differentially expressed genes in DPs from RAG2-K01/4 (KO) and TCRp-transduced RAG2-K01/4 (KOTCRb) cells.
- Fig. 6B shows names of gene ontology biological pathways that involve genes that are differentially up-regulated in Control WT DPs compared to RAG2-KO DPs. Highlighted are biological pathways that are relevant to cell survival and/or proliferation. Within the group of biological pathways that are involved in specific aspects of leukocyte regulation, the specific genes involved are also indicated.
- Fig. 7 (A-B) shows forced expression of TCRaP and TCRy5 chains in RAG2-KO CD4 + CD8 + DPs.
- Fig. 7A shows TCRap- and TCRy8-transduced DPs displayed higher cell numbers after 4 and 10 days of culture compared to dTomato- transduced DPs. From left to right are dTomato, TCRap- and TCRy8.
- Fig. 7B is a flow cytometric analysis of TCR-transduced RAG2-KO DP cells showing expression of the corresponding ab and gd TCRs, respectively, on the cell surface of T-lineage cells.
- the inventors differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) and showed that human RAG2-deficient developing T cells progress up to the CD4+CD8+ stage.
- RAG2-KO CD4+CD8+ double positive cells can be engineered to express TCRap and TCRy8 chains and that expression of a rearranged TCRp chain promotes cell survival and/or proliferation of developing human T-cells at the double positive stage.
- the disclosure provides a method of generating stem or progenitor cells unable to undergo T cell receptor (TCR) gene rearrangements, the method comprising:
- the method further comprises (b) isolating cells of the T cell lineage.
- the disclosure also provides a method of generating a cell of the T cell lineage comprising an unrearranged T cell receptor (TCR) gene locus, the method comprising: (a) culturing a sample comprising stem cells or progenitor cells, wherein the expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem or progenitor cells.
- TCR T cell receptor
- cells of the T cell lineage refers to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages.
- phenotypic characteristics can include expression of one or more proteins specific for T lineage commitment on cells or a precursor or progenitor thereof, or a physiological, morphological, functional, or immunological feature specific for a T cell.
- the cells of the T cell lineage are human cells.
- a cell or “the cell” includes a plurality of cells.
- isolated means that a cell has been separated or purified from cellular or biological material found with the cells in their native environment. It thus distinguishes the cells from how they exist in nature.
- the disclosure also provides a method of generating stem or progenitor cells unable to undergo B cell receptor (BCR) gene rearrangements, the method comprising:
- the method further comprises (b) isolating cells of the B cell lineage.
- the disclosure further provides a method of generating a cell of the B cell lineage comprising an unrearranged B cell receptor (BCR) gene locus, the method comprising: (a) culturing a sample comprising stem cells or progenitor cells, and (b) isolating cells of the B cell lineage, wherein the expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem or progenitor cells.
- BCR B cell receptor
- cells of the B cell lineage refers to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B-lineage on cells or a precursor or progenitor thereof, or a physiological, morphological, functional, or immunological feature specific for a B cell.
- the cells of the B cell lineage are CD20+ or CD19+ cells.
- the cells of the B cell lineage are Tumor-Infiltrating B Cells (TIBs).
- the cells of the B cell lineage are human cells.
- Cells of the T cell lineage may be (a) progenitor or precursor cells committed to the T cell lineage (“progenitor T cells” or “proT cells”, as described herein); (b) CD7+ immature T cells; (c) cells that have undergone CD4 or CD8 lineage commitment (e.g.
- CD4+CD8 /0 TCR mi cells (d) characterized by TCR gene rearrangement; (e) precursor thymocytes that are CD4+CD8+ double positive (DP); (f) CD4-CD8+ orCD4+CD8- and optionally TCR h ; (g) CD3+CD90+; (h) single positive (SP) cells that are CD4-CD8+ or CD4+CD8- and TCR' 1 '; (i) TCR-ab * and/or TCR-y6 + ; (j) characterized by expression of any of multiple nb chains (e.g. nb-3, -6, and 17a); or (k) mature and functional or activated T cells which may be characterized as TCR/CD3 h ', CD4-CD8+ or CD4+CD8-.
- TCR gene rearrangement (e) precursor thymocytes that are CD4+CD8+ double positive (DP); (f) CD4-CD8+ orCD4+CD
- a cell of the T cell lineage is a “progenitor T cell’ or
- proT cell The term “progenitor T cell” or “proT cell” as used herein means a T cell that is capable of maturing into a mature T cell or lymphocyte. In one embodiment, the pro T cell is a CD45+CD34+CD7+ proT cell.
- the progenitor T cell is a human progenitor T cell.
- Phenotypes of human progenitor T cells include CD34+CD7+ and CD7+CD5+CD1a-.
- a cell of the T cell lineage is a CD4 and CD8 double positive (DP) cell characterized by a CD4+CD8+ or CD4+CD8+CD3+ phenotype.
- a cell of the T cell lineage is a CD4 or CD8 single positive (SP) cell characterized by a CD4-CD8+, CD4+CD8- or CD4-CD8+CD3+, CD4+CD8-CD3+ phenotype.
- TCR T-cell receptor
- BCR B-cell receptor
- the TCR gene locus can comprise an alpha (a) chain and a beta (b) chain (encoded for example by genes TRA and TRB, respectively), or a gamma and delta (g/d) chains (encoded for example by genes TRG and TRD, respectively).
- TRA and TRB beta chain
- g/d gamma and delta chains
- the term “gene or protein required for V(D)J recombination” refers to a gene of protein that is required for correct rearrangement of the B- or T-cell receptor (BCR or TCR) gene loci.
- the gene or protein required for V(D)J recombination is a lymphocyte specific gene such as RAG1 or RAG2 or TdT.
- the gene or protein required for V(D)J recombination is a member of the non-homologous end joining (NHEJ) pathway for DNA repair.
- NHEJ non-homologous end joining
- the gene or protein required for V(D)J recombination is Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair crosscomplementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1 ; also known as Cernunnos or XRCC4-like factor (XLF)), Paralog of XRCC4 and XLF (PAXX), or the DNA polymerases l and m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair crosscomplementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- XLF Cernunnos or XRCC4-like factor
- PAXX Paralog of XRCC4 and XLF
- V(D)J recombination is RAG1 or RAG2.
- the V(D)J Recombination Activating Genes (RAG) 1 and 2 encode two essential DNA processing enzymes required for the rearrangement of the B- and T-cell receptor (BCR orTCR) gene loci (Schatz at al, 1989; Oettinger et al, 1990).
- BCR orTCR B- and T-cell receptor
- the inventors differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2- KO) and showed that human RAG2-deficient developing T-cells progress up to the CD4+CD8+ stage.
- a cell of the T cell lineage or B cell lineage may be generated by culturing a sample comprising stem cells or progenitor cells, wherein the expression and/or function of RAG1 and/or RAG2 in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells.
- RAG1 refers to V(D)J Recombination Activating Protein
- RAG1 is encoded by the RAG1 gene.
- RAG1 includes RAG1 from any species or source. The term also includes sequences that have been modified from any of the known published sequences of RAG1 proteins and genes. RAG1 orthe RAG1 gene may have any of the known published sequences for RAG1 which can be obtained from public sources such as GenBank.
- RAG1 is human RAG1. Examples of human amino acid sequences for RAG1 include GenBank accession no. AAQ13571.1. Examples of human nucleic acid sequences for RAG1 include include GenBank no. NM_001377277.1 (Gene ID: 5896). The aforementioned sequences are incorporated herein by reference.
- RAG2 refers to V(D)J Recombination Activating Protein (RAG) 2, which is encoded by the RAG2 gene.
- RAG2 includes RAG2 from any species or source. The term also includes sequences that have been modified from any of the known published sequences of RAG2 proteins and RAG2 genes. RAG2 or the RAG2 gene may have any of the known published sequences for RAG2 which can be obtained from public sources such as GenBank.
- RAG2 is human RAG2. Examples of the human amino acid sequences for RAG2 include GenBank accession no. AAH22397.1. Examples of human nucleic acid sequences for RAG2 include GenBank accession no. NMJD00536.4 (Gene ID: 5897). The aforementioned sequences are incorporated herein by reference.
- RAG1 expression includes both RAG1 protein expression and RAG1 gene expression.
- RAG2 expression includes both RAG2 protein expression and RAG2 gene expression.
- RAG1 function and “RAG2 function” refers to the biological activity of the RAG1 and RAG2, respectively.
- RAG1 and RAG2 function refers to the biological activity of the RAG1 and RAG2, respectively.
- BCR or TCR B cell receptor and T cell receptor
- V(D)J recombination (for example endogenous expression and/or function of RAG1 and/or RAG2) can be reduced or eliminated using any of a number of methods known in the art.
- Reduced or eliminated expression and/or function of a gene or protein required for V(D)J recombination may be accomplished by mutation of deletion of the corresponding gene sequence.
- Reduced or eliminated expression and/or function of RAG1 or RAG2 may be accomplished for example by mutation or deletion of RAG1 or RAG2 gene sequences.
- RAG1 or RAG2 gene sequences may be mutated or deleted from the genome of pluripotent stem cells using, for example, gene editing methods.
- RNA/DNA guided endonucleases e.g., Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9, Cpf1 , and Argonaute
- CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
- TALE Transcription Activator- Like Effector
- ZFN zinc finger nucleases
- meganucleases can be adapted for use in embodiments of the present disclosure.
- insertions or deletions are made by gene editing to cause a frame shift mutation, leading to gene knock out (i.e., lack of expression of a functional gene product).
- protocols used in the present invention in the context of RAG2 include transfection of an expression vector containing cassettes for GFP, Cas9 endonuclease, a CRISPR chimeric cDNA and a gRNA moiety that targets RAG 2.
- a RAG1 and/or RAG2 inhibitor is used to reduce endogenous RAG1 and/or RAG2 expression and/or function.
- the term “inhibitor” refers to an agent that reduces, decreases or otherwise blocks expression or activity of its target and includes any substance that is capable of inhibiting the expression or activity of the target and includes, without limitation, small molecules, antisense oligonucleotide molecules (antisense nucleic acid molecules), siRNAs or shRNAs, aptamers, proteins, antibodies (and fragments thereof), gene editing agents and other substances directed at the target expression or activity.
- Endogenous RAG1 and/or RAG2 expression and/or function can be reduced transiently or permanently (for example, by introducing gene mutations or deletions into the germline).
- wildtype refers to a cell which has normal (non- modified), endogenous expression levels of a gene or protein required for V(D)J recombination.
- wildtype refers to a cell which has normal (non-modified), endogenous expression levels of RAG1 and/or RAG2 genes or proteins.
- the wildtype cell is optionally a stem or progenitor cell.
- endogenous RAG1 expression and/or function is reduced by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell.
- the cell has no detectable endogenous RAG1 expression and/or function.
- endogenous RAG2 expression and/or function is reduced by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell.
- the cell has no detectable endogenous RAG2 expression and/or function.
- the stem or progenitor cell is a pluripotent stem cell.
- pluripotent stem cell refers to any stem cell having the potential to differentiate into all cell types of a human or animal body, not including extra- embryonic tissues. These stem cells include both embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs).
- ESCs embryonic stem cells
- iPSCs induced pluripotent cells
- the cells suitable for the method of the present invention include stem cells selected from iPSCs and ESCs.
- the pluripotent stem cells are human pluripotent stem cells (hPSCs) and they include human iPSCs (hiPSCs) and human ESCs (hESCs).
- embryonic stem cell or “ESC” as used herein refers to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germ line of a developing embryo.
- iPSC induced pluripotent stem cell
- somatic cells such as skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state.
- iPSCs are derived from T cells with a known or unknown TCR specificity (for example, T cells bearing TCRs with specificity against cancer).
- the stem or progenitor cell is a hematopoietic stem or progenitor cell (HSPC).
- the stem or progenitor cell is a CD34 + hematopoietic precursor cell, optionally a CD34 + hemogenic endothelial precursor cell that has been differentiated from an ESC or iPSC, or a CD34 + pre-hematopoietic cell differentiated from an ESC or pluripotent stem cell (PSC).
- PSC pluripotent stem cell
- Various differentiation protocols for obtaining CD34 + cells are known in the art.
- the stem cells or progenitor cells used to generate the cells of the T cell lineage may be obtained from the patient to be treated.
- Stem or progenitor cells may be obtained from any suitable source, including, without limitation, umbilical cord blood, embryos, embryonic tissue, fetal tissue, bone marrow and blood.
- a sample containing stem or progenitor cells is first depleted of non-stem cells or mature cells.
- Negative and positive selection methods known in the art may be used for enrichment of the stem or progenitor cells. For example, cells can be sorted based on cell surface antigens using a fluorescence activated cell sorter, or magnetic beads which bind cells with certain cell surface antigens. Negative selection columns can be used to remove cells expressing lineage specific surface antigens.
- a sample containing stem or progenitor cells is separated into lineage-negative (Lin ) and lineage position (Lin + ) fractions.
- the Lin- fraction can be sorted for CD34 + cells.
- the progenitor cells or stem cells may be cultured under suitable conditions to generate cells of the T cell lineage or B cell lineage. Methods of culturing progenitor cells or stem cells to generate cells of the T cell lineage or B cell lineage are known in the art.
- human pluripotent stem cells may be induced to differentiate as embryoid bodies, then CD34+ cells may be isolated by magnetic-assisted cell-sorting and placed in co-culture with OP9-DL4 cells to induce their differentiation towards the T cell.
- OP9-DL4 cells Such a protocol is described in Kennedy et al. 2012.
- the cells are cultured in the presence of one or more Notch ligands conjugated to a suspension support for a sufficient time to form cells of the T cell lineage.
- the progenitor cells or stem cells may be cultured on plates or in suspension in a bioreactor, optionally a closed or a closed, automated bioreactor.
- a bioreactor optionally a closed or a closed, automated bioreactor.
- Various bioreactors are known in the art and can include batch, fed batch or continuous bioreactors.
- An example of a continuous bioreactor is a continuous stirred-tank reactor model.
- concentrations of progenitor cells or stem cells in the culture are contemplated.
- the concentration of progenitor cells or stem cells in the culture may be anywhere from 1 to millions of cells per ml of media.
- One or more positive cytokines that promote commitment and differentiation of cells of the T cell lineage or B cell lineage may also be added to the culture.
- the cytokines may be human in origin, or may be derived from other species.
- the concentration of a cytokine in a culture is typically about 1-10ng/ml.
- the cytokines used herein are Flt-3-ligand and IL-7 and Stem Cell Factor.
- the cytokines may be used in combination with equal molar or greater amounts of a glycosaminoglycan such as heparin sulfate.
- the cytokines are commercially available or can be produced by recombinant DNA techniques and purified to various degrees. Some of the cytokines may be purified from culture media of cell lines by standard biochemical techniques.
- the additional molecule is a molecule that promotes T cell development (for example, promotes commitment and differentiation of cells of T cell lineage), also referred to herein as a “T cell co stimulatory molecule”.
- T cell co stimulatory molecule the inventors have shown that microbead- conjugated DL4 and VCAM1 cultured with HSPCs accelerated differentiation to the T cell lineage.
- the T cell co-stimulatory molecule is VCAM 1.
- VCAM1 refers to Vascular cell adhesion protein 1 also known as vascular cell adhesion molecule 1 (VCAM1) or cluster of differentiation 106 (CD106), a protein that in humans is encoded by the VCAM1 gene.
- VCAM 1 also includes a mutant or variant of a VCAM1.
- the T cell co-stimulatory molecule is a cytokine or chemokine (Stem Cell Factor, IL-7, CCL25, or CXCR4), Major Histocompatibility Complex (MHC) class I or class II, or co-stimulatory (CD80, CD86) molecule.
- the T cell co-stimulatory molecule comprises at least one protein tag.
- Various protein tags are known in the art and can be used for a number of purposes.
- the T cell co-stimulatory molecule comprises an Fc tag (also known as an Fc-fusion protein).
- the progenitor cells and stem cells may be cultured in culture media comprising conditioned media, non-conditioned media, or embryonic stem cell media.
- suitable conditioned media include IMDM, DMEM, or aMEM, conditioned with embryonic fibroblast cells (e.g. human embryonic fibroblast cells or mouse embryonic fibroblast cells), or equivalent media.
- suitable non-conditioned media include Iscove’s Modified Dulbecco’s Medium (IMDM), DMEM, or aMEM, or equivalent media.
- the culture media may comprise serum (e.g. bovine serum, fetal bovine serum, calf bovine serum, horse serum, human serum, or an artificial serum substitute) or it may be serum free.
- the culture conditions entail culturing the progenitor cells or stem cells for a sufficient period of time so that cells in the preparation form proT cells.
- the culture conditions entail culturing the progenitor cells or stem cells for a sufficient period of time so that cells in the preparation form mature T cells, for example mature SP T cells. It will be appreciated that the cells may be maintained for the appropriate amount of time required to achieve the desired cellular composition.
- the progenitor cells or stem cells are cultured for at least 6, 8, 10, 12, 14, 21 , 28, 35 or 42 days.
- the method further comprises engineering the cells to comprise a nucleic acid encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In another embodiment, the method further comprises engineering the cells to comprise a nucleic acid encoding a T cell receptor beta chain (TCRp).
- TCR T cell receptor
- CAR chimeric antigen receptor
- TCRp T cell receptor beta chain
- the cells may be engineered to comprise a nucleic acid encoding a TCR, CAR and/or TCRp at any time during the differentiation process.
- the stem or progenitor cells comprise a nucleic acid encoding a TCR, CAR and/or TCRp.
- the cells of the T cell lineage comprise a nucleic acid encoding a TCR, CAR and/or TCRp.
- the stem or progenitor cells and/or the cells of the T cell lineage express a TCR, CAR and/or TCRp.
- the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp alone.
- the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and do not comprise a nucleic acid encoding a TCR or CAR.
- the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and a nucleic acid encoding a CAR.
- the TCRp directs differentiation while the CAR can provide therapeutic efficacy.
- the method further comprises engineering the cells of the B-cell lineage to express a B cell receptor (BCR), B cell receptor beta chain (BCRp) and/or CAR.
- BCR B cell receptor
- BCRp B cell receptor beta chain
- the cells may be engineered to comprise a nucleic acid encoding a BCR, BCRp and/or CAR at any time during the differentiation process.
- the stem or progenitor cells comprise a nucleic acid encoding a BCR, BCRp and/or CAR.
- the cells of the B cell lineage comprise a nucleic acid encoding a BCR, BCRp and/or CAR.
- the stem or progenitor cells and/or the cells of the B cell lineage express a BCR, BCRp and/or CAR.
- the cells may be engineered by any method known in the art to comprise a nucleic acid encoding a TCR, TCRp, CAR, BCR or BCRp.
- the cells may be transformed with viral or non-viral vectors carrying a TCR, TCRp, CAR, BCR or BCRp.
- viral vectors include, but are not limited to, retroviruses (including lentivirus), adenoviruses and adeno-associated viruses.
- non-viral vectors include, but are not limited to, nude DNA, minicircle DNA vectors, liposomes, polymerizers and molecular conjugates.
- the TCR, BCR or CAR optionally confers specificity to an antigen such as a tumour associated antigen, a viral antigen or a self-antigen.
- a “tumor associated antigen” is an antigen produced by tumor cells. Examples of tumor associated antigens include, but are not limited to, alphafetoprotein (AFP), arcinoembryonic antigen (CEA), CA-125, MUC-1 , Epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), WT1 and NYES01.
- a “viral antigen” is an antigen encoded by a viral genome.
- examples of viral antigens include, but are not limited to EBV, CMV, HIV and SARS viral antigens.
- a “self-antigen” is an antigen that is produced by the subject.
- a selfantigen can be a tumor associated antigens.
- a T cell expressing a TCR or CAR conferring specificity to a self-antigen could be used for example to make regulatory T cells that block self-reactive T cells.
- RAG2-deficient (RAG2-KO) human pluripotent stem cell (hPSC) lines were generated RAG2-deficient (RAG2-KO) human pluripotent stem cell (hPSC) lines.
- the disclosure also provides an isolated stem or progenitor cell, wherein expression of at least one gene or protein required forV(D)J recombination, optionally RAG 1 and/or RAG2, in the stem cell or progenitor cell is reduced or eliminated compared to a wildtype stem cell or progenitor cell.
- the stem cell or progenitor cell is human cell.
- the stem cell is a pluripotent stem cell.
- the pluripotent stem cell is an embryonic stem cell or induced pluripotent stem cell (iPSC).
- RAG 1 or RAG2 may be accomplished by any one of a number of methods known in the art.
- the stem cell or progenitor cell comprises a mutated or deleted RAG1 and/or RAG2 gene sequence.
- insertions or deletions in the RAG1 and/or RAG2 gene sequences may cause a frame shift mutation, leading to gene knock out (i.e., lack of expression of a functional gene product).
- endogenous RAG1 expression and/or function is reduced in the stem cell or progenitor cell by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG1 expression and/or function. [00136] In another embodiment, endogenous RAG2 expression and/or function is reduced in the stem cell or progenitor cell by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG2 expression and/or function.
- the stem cell or progenitor cell further comprises a nucleic acid encoding a TCR, CAR, TCRp, BCR or BCRp.
- stem cell or progenitor cell expresses a TCR, CAR, TCRp, BCR or BCRp.
- the stem or progenitor cell comprises a nucleic acid encoding a TCRp and does not comprise a nucleic acid encoding a TCR or CAR.
- the disclosure further provides cells of the T cell lineage or B cell lineage generated by the methods, systems and kits described herein, or mitotic or differentiated cells that are progeny of the cells.
- the disclosure provides a “progenitor T cell’ or “proT cell” generated by the methods described herein.
- the progenitor T cell is a human progenitor T cell, for example a human progenitor T cell characterized by CD34+CD7+, CD7+CD5+CD1a- or CD45+CD34+CD7+.
- the disclosure also provides a double positive (DP) T-cell characterized by CD4+CD8+ or CD4+CD8+CD3+.
- the disclosure further provides a cell of the T-cell lineage that is a single positive (SP) cell characterized by CD4-CD8+, CD4+CD8- or CD8+CD3+, CD4+CD3+.
- the cells of the T cell lineage further comprise a nucleic acid encoding a TCR, CAR or TCRp. In another embodiment, the cells of the T cell lineage express a TCR, CAR or TCRp.
- the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and does not comprise a nucleic acid encoding a TCR or CAR.
- a cell of the T cell lineage generated by the methods described herein is engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
- TCR T cell receptor
- CAR chimeric antigen receptor
- the antigen is optionally a tumor associated antigen (TAA), viral antigen or self-antigen.
- T-cells engineered to express a TCR or a CAR to confer specificity to a TAA can be useful for treating conditions such as cancer.
- the cells of the B cell lineage further comprise a nucleic acid encoding a CAR, TCRp, BCR or BCRp. In another embodiment, the cells of the B cell lineage express a CAR, BCR or BCRp.
- a cell of the B cell lineage generated by the methods described herein is engineered with a B cell receptor (BCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
- BCR B cell receptor
- CAR chimeric antigen receptor
- the antigen is optionally a tumor associated antigen (TAA), viral antigen or self-antigen.
- TAA tumor associated antigen
- BCR BCR
- CAR CAR
- the present disclosure provides a pharmaceutical composition
- a pharmaceutical composition comprising isolated stem or progenitor cells or cells of the T cell lineage or B cell lineage generated by the methods described herein and a pharmaceutically acceptable diluent or carrier.
- compositions include, albeit not exclusively, solutions of the proT cells in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso- osmotic with physiological fluids.
- compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
- Other components that may be present in such compositions include water, surfactants (such as TweenTM), alcohols, polyols, glycerin and vegetable oils, for example.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
- compositions may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.
- Pharmaceutical compositions also include cyropreservative solutions.
- cells of the T cell lineage generated by the methods described herein are cryopreserved in appropriate media, for example pharmaceutically acceptable or GMP-grade media and optionally formulated for administration to a subject in need thereof.
- Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
- suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
- DOTMA N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride
- DOPE diolesylphosphotidyl-ethanolamine
- liposomes Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
- compositions can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.
- solutions of the pro-T cells described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.
- the cells of the T cell lineage or B cell lineage are present in an amount effective for treating a disease state in a subject need thereof.
- the cell of the T cell lineage is present in an amount effective to enhance hematopoietic progenitor cell engraftment in a subject in need thereof.
- the composition further comprises cells of the T cell lineage, or tissue for transplantation.
- the tissue comprises a thymus.
- the tissue comprises an organ.
- Kits Stem or progenitor cell as described herein may be prepared and packaged in kits for use in generating cells of the T cell lineage or cells of the B cell lineage.
- kits for producing cells of the T cell lineage or cells of the B cell lineage comprising a stem or progenitor cell, wherein the expression of RAG1 and/or RAG2 in the stem cell or progenitor cell is reduced or eliminated compared to wildtype stem cell or progenitor cells.
- the kit further comprises culture media for culturing a sample comprising stem cells or progenitor cells for producing cells of the T cell lineage or cells of the B cell lineage.
- culture media include conditioned media, non-conditioned media, or embryonic stem cell media.
- the culture media may comprise serum (e.g. bovine serum, fetal bovine serum, calf bovine serum, horse serum, human serum, or an artificial serum substitute) or it may be serum free.
- the kit further comprises one or more additional molecules.
- the additional molecule is a molecule that promotes T cell development (for example, promotes commitment and differentiation of cells of T cell lineage), also referred to herein as a “T cell co-stimulatory molecule”.
- the T cell co-stimulatory molecule is VCAM1.
- the media optionally includes one or more cytokines that promote commitment and differentiation of cells of the T cell lineage or cells of the B cell lineage.
- the cytokines may be human in origin, or may be derived from other species.
- the concentration of a cytokine in a culture is typically about 1-10ng/ml.
- the following are representative examples of cytokines that may be employed in the present application: all members of the Flt-3-ligand, and interleukin-7 (IL-7) and Stem Cell Factor.
- the cytokines used herein are Flt-3-ligand and IL-7 and Stem Cell Factor.
- the cytokines may be used in combination with equal molar or greater amounts of a glycosaminoglycan such as heparin sulfate.
- the cytokines are commercially available or can be produced by recombinant DNA techniques and purified to various degrees. Some of the cytokines may be purified from culture media of cell lines by standard biochemical techniques.
- the kit comprises one or more containers for the within-described reagents.
- Printed instructions providing guidance in the use of the reagent(s) may also be included in the kit, in various embodiments.
- the term “instructions” or “instructions for use” typically includes a tangible expression describing the cells, culturing time periods, temperature, media conditions, and the like.
- the instructions describe a method comprising culturing a sample comprising stem cells or progenitor cells.
- T cells and B cells engineered to recognize specific antigens have wide ranging therapeutic applications.
- the disclosure provides method of treating a disease or condition in a subject comprising:
- stem cells or progenitor cells are engineered to comprise at least one of a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a B cell receptor (BCR) that confers specificity to an antigen.
- TCR T cell receptor
- CAR chimeric antigen receptor
- BCR B cell receptor
- the disclosure also provides a method of treating a disease or condition in a subject comprising:
- TCR T cell receptor
- CAR chimeric antigen receptor
- the disclosure also provides a use of cells of the T cell lineage, generated by the methods described herein for treating a disease or condition in a subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confer specificity to an antigen.
- TCR T cell receptor
- CAR chimeric antigen receptor
- the disclosure further provides a use of cells of the T cell lineage, generated by the methods described herein for preparation of a medicament for treating a disease or condition in a subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
- the disclosure also provides cells of the T cell lineage, generated by the methods described herein for use in treating a disease or condition in a subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
- TCR T cell receptor
- CAR chimeric antigen receptor
- the disclosure also provides a method of treating a disease or condition in a subject comprising:
- the cells of the B cell lineage are engineered with at least one B cell receptor (TCR) to confer specificity to an antigen.
- TCR B cell receptor
- the disclosure also provides a use of cells of the B cell lineage, generated by the methods described herein for treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen.
- BCR B cell receptor
- the disclosure further provides a use of cells of the B cell lineage, generated by the methods described herein for preparation of a medicament for treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen.
- the disclosure also provides cells of the B cell lineage, generated by the methods described herein for use in treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen.
- BCR B cell receptor
- the antigen is optionally a tumor associated antigen (TAA), a viral antigen or a self-antigen.
- TAA tumor associated antigen
- the antigen is a TAA and the disease is cancer.
- the phrase "effective amount” or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex, weight of the subject. The amount of a given cell preparation that will correspond to such an amount will vary depending upon various factors. Such as the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. An “effective amount” will preferably be an amount effective for the cell of the T cell lineage to engraft the subject being treated.
- treating means an approach for obtaining beneficial or desired results, including clinical results.
- beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
- Treating and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
- Treatment as used herein also includes prophylactic treatment.
- subject as used herein means any member of the animal kingdom and is preferably a human.
- hESC Maintenance Human ESCs (H1 ; WiCell Research Institute, Madison, Wl) were maintained and expanded on plates coated with Matrigel (Corning, NY, USA) in TeSR-E8 medium (STEMCELL Technologies, Vancouver, Canada). Cells were passaged by non-enzymatic dissociation using 0.5 mM EDTA.
- cellular lysates were prepared by incubating the cells in lysis buffer (50 mM Tris-HCI, pH7.5, 150 mM NaCI, 0.5% NP-40, 2 mM EDTA) containing protease inhibitor cocktail (Roche) for 20 min at 4°C, followed by centrifugation at 14000*g for 15 min at 4°C. Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore, Louis, MO) and probed with anti-RAG2 antibody (Abeam - Ab95955; 1 :1000 dilution) overnight at 4°C followed by incubation with secondary antibody.
- lysis buffer 50 mM Tris-HCI, pH7.5, 150 mM NaCI, 0.5% NP-40, 2 mM EDTA
- protease inhibitor cocktail Roche
- the anti-RAG2 antibody used was a rabbit polyclonal made against a recombinant fragment corresponding to amino acids 271-519 of human RAG2 (Abeam), which are well beyond the gRNA targeting site. Immunoreactive bands were visualized using western blotting Luminol reagent (Thermo). PBMCs from a T-ALL patient were used as positive control (Bories et al, 1991).
- embryoid bodies were harvested and dissociated into single-cells using Collagenase type IV and trypsin-EDTA as previously described (Kennedy et al, 2012), and positively selected using a MACS column (Miltenyi Biotec) with anti-CD34 PE-conjugated antibody (BD Biosciences) and anti-PE microbeads.
- MACS column Miltenyi Biotec
- anti-CD34 PE-conjugated antibody BD Biosciences
- anti-PE microbeads anti-PE microbeads.
- the cell yield and purity of the positive selection was assessed pre- and post- MACS by flow cytometry.
- OP9-DL4-7FS Co-Culture and Differentiation.
- OP9-DL4 cells expressing hlL-7, hFLT3-L, and hSCF (7FS) were generated and grown in a-MEM containing 10-20% FBS (Gibco), 1% Pen-Strep (Life Technologies), and phospho- ascorbic acid (Sigma-Aldrich) at 37°C, 5% CO2.
- FBS Gibco
- Pen-Strep Life Technologies
- phospho- ascorbic acid Sigma-Aldrich
- MACS-purified CD34 + cells were counted and seeded at a density of 5-20 x10 4 cells per 6-well plate, and differentiated by coculture with OP9-DL4-7FS cells in OP9 medium at 37°C, 5% CO2, and cell were passaged and co-cultured with fresh OP9-DL4-7FS cells every ⁇ 5 days.
- Retroviral Transduction PG13 cell lines stably expressing empty vector dTomato, TCRa-dTomato, or TCRp-dTomato were grown towards 70% confluency, at which point the media was switched to a-MEM with 15% FBS and 1% Pen-Strep to condition the supernatant for 48 h before transducing OP9-DL4-7FS/T-cell cultures.
- RNA-Seq Analysis of Control and RAG2-KO in vitro Derived CD4 + CD8 + cells were resuspended in flow cytometry buffer containing DAPI, data was collected using LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo version 9.7.6. For intracellular staining, cells were fixed and permeabilized using Fixation/Permeabilization kit with GolgiPlugTM (BD Biosciences) as per manufacturer’s instructions. [00187] RNA-Seq Analysis of Control and RAG2-KO in vitro Derived CD4 + CD8 + cells.
- FIG. 1A CRISPR-Cas9 gene editing was used to target exon 3 of the RAG2 gene.
- hPSCs were transfected with a plasmid encoding the RAG2-targeting guide RNA, the Cas9 enzyme, and green fluorescent protein (GFP).
- GFP + hPSCs were single-cell sorted and cultured. After expanding individual clones, two clones were identified that contained unique insertion-deletions with bi-allelic mutations (KO-1 and KO-4) (Fig. 1A).
- Western blot analysis was performed, which confirmed the absence of detectable RAG2 protein in both KO-1 and -4 derived T-lineage cells (Fig. 1B).
- CD34 expression was analyzed after 8 days of embryoid- body differentiation cultures (Kennedy et al, 2012).
- Control WT, RAG2-KO-1 and -4 hPSCs gave rise to similar frequencies of hemogenic endothelial CD34 + cells, which could be further enriched by magnetic-assisted cell sorting (MACS) (Fig. 1E).
- MCS magnetic-assisted cell sorting
- T-cell development from RAG2-KO hPSCs CD34 + hemogenic endothelial cells were MACS-enriched and cultured with OP9-DL4 cells, expressing human IL-7, FLT3-ligand and stem cell factor (7FS), to induce T-cell differentiation.
- Control WT, RAG2-KO-1 and -4 cells proceeded along the T-cell lineage, as marked by expression of both CD7 and CD5 (Fig. 2A). All three groups reached the CD4 + intermediate single positive (ISP) stage by day 15 and displayed intracellular CD3 expression by day 20 (Fig. 2A).
- ISP intermediate single positive
- RAG2-KO hPSC-derived CD34 + cells were cultured on OP9-DL4-7FS cells for 24 days, and DP cells were retrovirally-transduced with an empty vector (dTomato), a rearranged TCRa chain (TRA 1383 ⁇ ), or a rearranged TCRp chain (TRB 1383i) (Fig. 3A).
- Transduced RAG-KO cells were sorted for CD7 + CD5 + CD4 + CD8 + DP cells, and placed back on OP9-DL4-7FS cells for an additional 10 days to assess for cell survival and expansion (Fig. 3A). Both dTomato- and TCRa-transduced DPs showed similar cell numbers after 10 days of culture (Fig. 3B).
- TCRp-transduced DPs displayed significantly higher cell numbers after 10 days of culture compared to dTomato- and TCRa-transduced DPs (Fig. 3B). Without being bound by theory, this suggests that, in contrast to a TCRa chain, expression of a rearranged TCRp chain promotes cell survival and/or proliferation of developing human T-cells at the DP stage.
- Control WT, RAG2-KO, and RAG2-KO TCRp-transduced CD4 + CD8 + DP cells were sorted for RNA sequencing (RNA-Seq) analysis.
- Control WT DP cells expressed a large set of TCRa, TCRp, TCRy, and TCR6 genes that were absent in RAG2-KO control-transduced and RAG-KO TCRp- transduced DP cells, with the notable exception of some TCR genes, including the 1383i TCRp used in the transduction and a few TCRa genes, likely the result of germline transcripts induced by the b-selection signals, as seen in mice (Villey et al, 1997) (Table 1, and Fig. 4A).
- RAG2-KO hPSCs were generated to assess the role of TCRp during human T-cell development.
- Lack of RAG 1/2 in mice results in a definitive block at the CD44 CD25 + DN3 stage, as it is well documented that RAG1/2-mediated TCRp rearrangement controls the transition from the DN3 to the CD44 CD25- DN4 and DP stages (von Boehmer et al, 1999; Michie and Zuniga-Pflucker, 2002).
- mice expression of a preTCR in DN3 cells leads to expression of Bmi-1 , which represses the expression of the cell cycle inhibitor Cdkn2a. Repression of Cdkn2a is required for preTCR-induced cell proliferation and the DN3-DP transition (Miyazaki et al, 2008).
- this study reveals the unexpected timing required for TCRb-mediated b-selection in developing human T-cells.
- RAG2-KO hPSC-derived CD34 + cells were cultured on OP9-DL4-7FS cells for 21 days, and DP cells were retrovirally-transduced with an empty vector (dTomato), rearranged TCRa ⁇ chains (1383i-TCR) (Roszkowski et al., 2003), or rearranged TCRy8 chains (3C2-TCR) (Benveniste et al., 2018) (Fig. 7).
- Transduced RAG-KO cells were sorted for CD7 + CD5 + CD4 + CD8 + DP cells, and placed back on OP9- DL4-7FS cells for an additional 4 and 10 days to assess for cell survival and expansion (Fig. 7A).
- TCRa ⁇ - and TCRy8-transduced DPs displayed higher cell numbers after 4 and 10 days of culture compared to dTomato-transduced DPs (Fig. 7A), with TORab- transduced cells showing a much higher fold expansion than TCRy5-transduced cells, and akin to what was seen with TCRb-transduced DP cells (Fig. 3).
- TCR-transduced RAG2-KO DP cells showed the expression of the corresponding ab and gd TCRs, respectively, on the cell surface of T-lineage cells (Fig. 7B).
- CRISPR-Cas9 gene editing is used to target each of the following genes: Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1 ; also known as Cernunnos orXRCC4-like factor (XLF)), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
- DNA-PK DNA-dependent protein kinase
- XRCC4 X-ray repair cross-complementing protein 4
- NHEJ1 non-homologous end-joining factor 1
- XLF Cernunnos orXRCC4-like factor
- PAXX Paralog of XRCC4 and XLF
- DNA polymerase l DNA polymerase m.
- hPSCs are transfected with a plasmid encoding the gene targeting guide RNA, the Cas9 enzyme, and green fluorescent protein (GFP).
- GFP + hPSCs are single-cell sorted and cultured.
- KO hPSCs are injected into immunodeficient mice, and histological analysis reveals KO teratoma formation with all three germ layers, indicating that KO hPSCs retain key features of pluripotency.
- CD34 expression is analyzed after 8 days of embryoid-body differentiation cultures (Kennedy et al, 2012). Control WT and KO hPSCs give rise to similar frequencies of hemogenic endothelial CD34 + cells.
- T-cell development from KO hPSCs CD34 + hemogenic endothelial cells are MACS-enriched and cultured with OP9-DL4 cells, expressing human IL-7, FLT3-ligand and stem cell factor (7FS), to induce T-cell differentiation. After 10 days of culture, Control WT and KO cells proceed along the T-cell lineage, as marked by expression of both CD7 and CD5. All three groups reach the CD4 + intermediate single positive (ISP) stage by day 15 and display intracellular CD3 expression by day 20. After 24 days of culture, the majority of cells from Control WT and KO groups are CD7 + CD5 + By 29-34 days of culture, Control WT and KO cells all reach the CD4 + CD8 + DP stage.
- ISP intermediate single positive
- KO hPSC- derived CD34 + cells are cultured on OP9-DL4-7FS cells for 24 days, and DP cells are retrovirally-transduced with an empty vector (dTomato), a rearranged TCRa chain (TRA 1383i), or a rearranged TCRp chain (TRB 1383i).
- Transduced KO cells are sorted for CD7 + CD5 + CD4 + CD8 + DP cells, and placed back on OP9-DL4-7FS cells for an additional 10 days to assess for cell survival and expansion.
- Both dTomato- and TCRa-transduced DPs show similar cell numbers after 10 days of culture. However, TCRp-transduced DPs display significantly higher cell numbers after 10 days of culture compared to dTomato- and TCRa-transduced DPs.
- KO SP T cells Generation of KO SP T cells.
- KO hPSCs engineered with one or more of a rearranged TCRpchain (TRB 1383i), a CAR or a TCR are differentiated into CD34+ cells and subsequently CD4+CD8+ DP cells as above or in stromal cell-free conditions (see for example WO2019157597A1 , the contents of which are incorporated by reference in their entirety).
- Generated CD4+CD8+ DP cells are further differentiated into CD4-CD8+ SP and/or CD8-CD4+ SP T cells.
- KO DP cells engineered with TCRp successfully progress to SP T cells in comparison with KO-only cells.
- Table 2 List of genes significantly up-regulated (p ⁇ 0.05) in Control WT DPs compared to RAG2-KO-4 DPs, or vice versa, as indicated: (NA: non-assigned TCR gene names).
- Table 3 List of genes significantly up-regulated (p ⁇ 0.05) in thymus DNs (CD34+CD7+CD1a+CD4-CD8-) compared to thymus DPs (CD4+CD8+), or vice versa from Casero et al., 2015 (18), as indicated: (NA: non-assigned TCR gene names).
- V(D)J recombination activating gene RAG-1. Cell 59: 1035-1048.
- Beta-selection is associated with the onset of CD8beta chain expression on
- CD4(+)CD8alphaalpha(+) pre-T cells during human intrathymic development CD4(+)CD8alphaalpha(+) pre-T cells during human intrathymic development.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A method of generating stem cells that are unable to undergo T cell receptor (TCR) or B cell receptor (BCR) gene rearrangements is provided. In particular, methods, compositions and kits for use in generating cells of the T cell lineage or B cell lineage comprising an unrearranged TCR gene locus or BCR gene locus, respectively, are 5 provided. In one embodiment, the cells are further engineered to express a TCR, bCR or CAR conferring specificity to an antigen of interest. Cells, compositions, kits and uses thereof are also provided.
Description
Title: STEM CELLS COMPRISING AN UNREARRANGED T CELL RECEPTOR (TCR) GENE LOCUS AND METHODS OF USE THEREOF
Cross-Reference to Related Application
[0001] This application claims the benefit of priority to United States Provisional
Application No. 63/178,990 filed April 23, 2021 , the contents of which are incorporated herein by reference in their entirety.
Field
[0002] The application relates to stem cells that are unable to undergo T cell receptor (TCR) or B cell receptor (BCR) gene rearrangements. In particular, the application relates to methods, compositions and kits for use in generating cells of the T cell lineage or B cell lineage comprising an unrearranged TCR gene locus or BCR gene locus, respectively.
Background
[0003] The ability of T cells to specifically recognize antigens is accomplished through the expression of specific T cell receptors encoded by the uniquely rearranged genomic loci of the TCRa and b chains. T cell development is predicated on the successful rearrangement of the T cell receptor (TCR) gene loci, which encode for antigen-specific receptors.
[0004] The V(D)J Recombination Activating Genes (RAG) 1 and 2 are two essential DNA processing enzymes required for the rearrangement of the B cell receptor and T cell receptor gene loci (Schatz at al., 1989; Oettinger et al, 1990). RAG 1/2 initiate V(D)J recombination by forming a complex that first recognizes and binds to recombination signal sequences (RSS) found adjacent to each V, D, and J gene segment. Upon formation of a synapse with another RSS, the RAG complex induces a double-strand DNA break, which is repaired by non-homologous end-joining process (Jones and Gellert, 2004; Smith et al, 2019). This ultimately results in the imperfect joining of different V, D, and J gene segments to potentially generate millions of different antigen receptors from a few hundred V(D)J segments.
Summary
[0005] The inventors differentiated human pluripotent stem cells with a
CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) and showed that human RAG2-deficient developing T cells progress up to the CD4+CD8+ double positive stage.
The inventors also showed that expression of a rearranged TCRp chain promotes cell survival and/or proliferation of developing human T-cells at the double positive stage.
[0006] Accordingly, the disclosure provides a method of generating stem or progenitor cells unable to undergo T cell receptor (TCR) gene rearrangements (TCR), the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells. [0007] In one embodiment, the method further comprises (b) isolating cells of the T cell lineage.
[0008] In another embodiment, the at least one gene or protein required for
V(D)J recombination is RAG1 and/or RAG2.
[0009] In another embodiment, the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[0010] In another embodiment, the stem cells are pluripotent stem cells. Optionally, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPSCs).
[0011] In another embodiment, the stem cells or progenitor cells are human cells.
[0012] In another embodiment, the cells of the T cell lineage are progenitor T (proT) cells, optionally CD45+CD34+CD7+ progenitor T (proT) cells.
[0013] In another embodiment, the cells of the T cell lineage are CD4+CD8+ double positive cells or CD4+CD8+CD3+ double positive cells.
[0014] In another embodiment, the cells of the T cell lineage are CD8+CD3+ single positive cells or CD4+CD3+ single positive cells. [0015] In another embodiment, the method further comprises engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise at least one of
a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
[0016] In another embodiment, the stem cells or progenitor cells or the cells of the T cell lineage express the at least one of a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
[0017] In another embodiment, the method further comprises engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise a nucleic acid encoding a TCRp chain. In a further embodiment, the stem cells or progenitor cells or the cells of the T cell lineage that comprise a nucleic acid encoding a TCRp chain do not comprise a nucleic acid that encodes a TCRp chain or a chimeric antigen receptor (CAR).
[0018] In another embodiment, the stem cells or progenitor cells or the cells of the T cell lineage express a TCRp chain.
[0019] In another embodiment, the stem cells or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp chain and a nucleic acid encoding a CAR.
[0020] In another embodiment, the TCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
[0021] The disclosure also provides a cell of the T cell lineage, wherein the cell is generated by a method described herein.
[0022] In one embodiment, the cell of the T cell lineage is a CD4+CD8+ double positive cell or a CD4+CD8+CD3+ double positive cell.
[0023] In another embodiment, the cell is a CD45+CD34+CD7+ progenitor T cell, CD8+CD3+ single positive cell or CD4+CD3+ single positive cell.
[0024] The disclosure also provides a stem or progenitor cell, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cell or progenitor cell is reduced or eliminated compared to a wild-type stem cell or progenitor cell.
[0025] In one embodiment, the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
[0026] In another embodiment, the at least one gene or protein required for
V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent
protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[0027] In another embodiment, the stem or progenitor cell further comprises at least one of a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
[0028] In another embodiment, the stem or progenitor cell further comprises a nucleic acid encoding a TCRp chain. In a further embodiment, the stem or progenitor cell that comprises a nucleic acid encoding a TCRp chain does not comprise a nucleic acid that encodes a TCRp chain or a chimeric antigen receptor (CAR).
[0029] In another embodiment, the stem cell is a pluripotent stem cell, optionally an embryonic stem cell or induced pluripotent stem cell (iPSC).
[0030] In another embodiment, the stem cell or progenitor cell is a human cell.
[0031] The disclosure also provides a use of a stem or progenitor cell as described herein for generating cells of the T cell lineage.
[0032] The disclosure also provides a kit comprising (i) a stem or progenitor cell as described herein and (ii) instructions for use of the stem or progenitor cell as described herein for generating cells of the T cell lineage.
[0033] The disclosure further provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells or progenitor cells to a subject in need thereof, wherein the stem cells or progenitor cells are engineered to comprise at least one of a nucleic acid encoding a T cell receptor (TCR) and a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[0034] The disclosure also provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the T cell lineage wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the T cell lineage to a subject in need thereof, wherein the stem cells or progenitor cells or the cells of the T cell lineage are engineered to comprise at least one of a nucleic acid encoding a T cell receptor (TCR) and a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[0035] In one embodiment, the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
[0036] In another embodiment, the at least one gene or protein required for
V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[0037] In another embodiment, the disease is cancer and the antigen is a tumor- associated antigen.
[0038] The disclosure also provides a method of generating stem or progenitor cells unable to undergo T cell receptor (BCR) gene rearrangements, the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells.
[0039] In one embodiment, the method further comprises (b) isolating cells of the B cell lineage.
[0040] In another embodiment, the at least one gene or protein required for
V(D)J recombination is RAG1 and/or RAG2.
[0041] In another embodiment, the at least one gene or protein required for
V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent
protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[0042] In another embodiment, the stem cells are pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells (iPSCs).
[0043] In another embodiment, the stem cells or progenitor cells are human cells.
[0044] In another embodiment, the cells of the B cell lineage are CD20+ or
CD19+ cells.
[0045] In another embodiment, the cells of the B cell lineage are Tumor-
Infiltrating B Cells (TIBs).
[0046] In another embodiment, the method further comprises engineering the stem cells or progenitor cells or the cells of the B cell lineage to comprise at least one of a nucleic acid encoding a B cell receptor (BCR), a BCRp chain and a chimeric antigen receptor (CAR).
[0047] In another embodiment, the stem cells or progenitor cells or the cells of the B cell lineage express the B cell receptor (BCR), the BCRp chain or the chimeric antigen receptor (CAR).
[0048] In another embodiment, the BCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
[0049] The disclosure also provides a cell of the B cell lineage, wherein the cell is generated by a method as described herein.
[0050] In one embodiment, the cell of the B cell lineage is a CD20+ or CD19+ cell.
[0051] In another embodiment, the cell of the B cell lineage is a Tumor-Infiltrating
B Cell (TIB).
[0052] The disclosure also provides a use of a stem or progenitor cell as described herein for generating cells of the B cell lineage.
[0053] The disclosure also provides a kit comprising (i) a stem or progenitor cell as described herein and (ii) instructions for use of the stem or progenitor cell as described herein for generating cells of the B cell lineage.
[0054] The disclosure also provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the stem cells or progenitor cells to a subject in need thereof, wherein the stem cells or progenitor cells are engineered to comprise a nucleic acid encoding a B cell receptor (BCR) that confers specificity to an antigen.
[0055] The disclosure also provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the B cell lineage wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the B cell lineage to a subject in need thereof, wherein the stem cells or progenitor cells orthe cells of the B cell lineage are engineered to comprise a nucleic acid encoding a B cell receptor (BCR) that confers specificity to an antigen.
[0056] In one embodiment, the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
[0057] In another embodiment, the at least one gene or protein required for
V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[0058] In another embodiment, the disease is cancer and the antigen is a tumor- associated antigen.
[0059] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the application are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.
Brief description of the drawings
[0060] Embodiments of the disclosure will now be described in relation to the drawings in which:
[0061] Fig. 1 (A-E) shows generation and characterization of RAG2-KO hESC lines. Fig. 1A shows Sanger Sequencing of genomic DNA of CRISPR/Cas9- targeted RAG2 of clones 1 and 4. The altered allele is depicted in chromatograph and sequence above it, while alignment of the altered and WT alleles are shown below. Fig. 1B shows an immunoblot for RAG2 protein expression of in v/fro-derived T-lineage cells from RAG2-KO clones (1 and 4) and Control WT hESCs, or Control PBMCs obtained from a T- ALL patient. GAPDH serves as a loading control. Fig. 1C shows immunofluorescence of RAG2-KO hESC clones for pluripotency markers. The scale bar corresponds to 100 pm. Fig. 1 D shows immunohistochemistry of RAG2-KO hESC clones against ectoderm (neural tissue), mesoderm (cartilage), and endoderm (glandular tissue) lineages of a teratoma formation assay. Fig. 1 E shows CD34+ hemogenic endothelial cell yield and enrichment after 8 days of embryoid body differentiation from Control WT, RAG2-KO-1 , and RAG2- KO-4 hESCs. (n=3 of three independent experiments).
[0062] Fig. 2 (A-B) shows T cell development from Control WT and RAG2-
KO hESC lines. Fig. 2A shows a representative flow cytometry analysis of Control WT and RAG2-KO (clones 1 and 4) hPSC-derived T-lineage cells from d8 EBs + 10-34d OP9-DL4-7FS co-cultures, as indicated. Cells were pre-gated for DAPI CD45T (n=5 of five independent experiments). Fig. 2B shows a cell numbers of Control WT and RAG2- KO (clones 1 and 4) hPSC-derived T-lineage cells per 10,000 input of CD34+ cells after indicated number of days of culture on OP9-DL4-7FS. (n=3 of three independent experiments).
[0063] Fig. 3 (A-B) shows forced expression of a rearranged TCRp chain in
RAG2-KO CD4+CD8+ DP cells results in cell expansion. Fig. 3A shows a schematic of the experimental approach. Fig. 3B shows cell counts of RAG2-KO DPs (clones 1
and 4) retrovirally-transduced with empty vector (dTomato), TCR alpha chain (TRA 1383i), or TCR beta chain (TRB 1383i) per 10,000 input of CD34+ cells. Transduced T- lineage cells (dTomato+) were first sorted for CD7+CD5+CD4+CD8+ double positive cells and then re-cultured on OP9-DL4-7FS for 10 days. (n=3 of three independent experiments).
[0064] Fig. 4 (A-B) shows TCR gene expression and CD4+CD8+ signature gene expression. Fig. 5A shows a heatmap analysis of specific TCR genes expressed in Control WT DPs but absent in RAG2-K01/4 DPs and TCRp-transduced RAG2-K01/4 DPs. Fig. 5B shows expression of CD4+CD8+ signature genes (as determined by thymus DP signature genes shown in Table 3) in RAG2-KO, TCRp-transduced RAG2-KO, and umbilical cord blood (UCB) derived DPs. (n=2 for Control WT, RAG2-KO-1, RAG2-KO-4, and UCB, and n=1 for RAG2-KO-1 TCRp-transduced and RAG2-KO-4 TCRp-transduced of one independent experiment).
[0065] Fig. 5 (A-B) shows transcriptomic analysis of Control WT, RAG2-
KO, and RAG2-KO TCRp-transduced CD4+CD8+ DP cells. RNA-seq analysis of Control WT, RAG2-K01/4, and TCRp+ RAG2-K01/4 CD4+CD8+ DPs. Shown are genes that are differentially highly expressed in Control WT DPs compared to RAG2-K01/4 DPs (Fig. 5A) and differentially highly expressed in RAG2-K01/4 DPs compared to Control WT DPs (Fig. 5B). (n=2 for Control WT, RAG2-KO-1, and RAG2-KO-4, and n=1 for RAG2-KO-1 TCRp-transduced and RAG2-KO-4 TCRp-transduced of one independent experiment).
[0066] Fig. 6 (A-B) shows an analysis of proliferation and differentiation genes and biological pathways. Fig. 6A is a heatmap analysis of differentially expressed genes in DPs from RAG2-K01/4 (KO) and TCRp-transduced RAG2-K01/4 (KOTCRb) cells. Fig. 6B shows names of gene ontology biological pathways that involve genes that are differentially up-regulated in Control WT DPs compared to RAG2-KO DPs. Highlighted are biological pathways that are relevant to cell survival and/or proliferation. Within the group of biological pathways that are involved in specific aspects of leukocyte regulation, the specific genes involved are also indicated.
[0067] Fig. 7 (A-B) shows forced expression of TCRaP and TCRy5 chains in RAG2-KO CD4+CD8+ DPs. Fig. 7A shows TCRap- and TCRy8-transduced DPs displayed higher cell numbers after 4 and 10 days of culture compared to dTomato- transduced DPs. From left to right are dTomato, TCRap- and TCRy8. Fig. 7B is a flow
cytometric analysis of TCR-transduced RAG2-KO DP cells showing expression of the corresponding ab and gd TCRs, respectively, on the cell surface of T-lineage cells.
Detailed Description
[0068] As described above, the inventors differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) and showed that human RAG2-deficient developing T cells progress up to the CD4+CD8+ stage. The inventors also showed that RAG2-KO CD4+CD8+ double positive cells can be engineered to express TCRap and TCRy8 chains and that expression of a rearranged TCRp chain promotes cell survival and/or proliferation of developing human T-cells at the double positive stage.
I. Method for generating cells
[0069] Accordingly, the disclosure provides a method of generating stem or progenitor cells unable to undergo T cell receptor (TCR) gene rearrangements, the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells. In one embodiment, the method further comprises (b) isolating cells of the T cell lineage.
[0070] The disclosure also provides a method of generating a cell of the T cell lineage comprising an unrearranged T cell receptor (TCR) gene locus, the method comprising: (a) culturing a sample comprising stem cells or progenitor cells, wherein the expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem or progenitor cells.
[0071] The term “cells of the T cell lineage” refers to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T lineage commitment on cells or a precursor or progenitor thereof, or a physiological, morphological, functional, or immunological feature specific for a T cell.
[0072] In one embodiment, the cells of the T cell lineage are human cells.
[0073] As used therein, the term “a cell” or “the cell” includes a plurality of cells.
[0074] As used therein, the term “isolated” means that a cell has been separated or purified from cellular or biological material found with the cells in their native environment. It thus distinguishes the cells from how they exist in nature.
[0075] The disclosure also provides a method of generating stem or progenitor cells unable to undergo B cell receptor (BCR) gene rearrangements, the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells. In one embodiment, the method further comprises (b) isolating cells of the B cell lineage.
[0076] The disclosure further provides a method of generating a cell of the B cell lineage comprising an unrearranged B cell receptor (BCR) gene locus, the method comprising: (a) culturing a sample comprising stem cells or progenitor cells, and (b) isolating cells of the B cell lineage, wherein the expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem or progenitor cells.
[0077] The term “cells of the B cell lineage” refers to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B-lineage on cells or a precursor or progenitor thereof, or a physiological, morphological, functional, or immunological feature specific for a B cell. In another embodiment, the cells of the B cell lineage are CD20+ or CD19+ cells. In another embodiment, the cells of the B cell lineage are Tumor-Infiltrating B Cells (TIBs).
[0078] In one embodiment, the cells of the B cell lineage are human cells.
[0079] Cells of the T cell lineage may be (a) progenitor or precursor cells committed to the T cell lineage (“progenitor T cells” or “proT cells”, as described herein); (b) CD7+ immature T cells; (c) cells that have undergone CD4 or CD8 lineage commitment (e.g. CD4+CD8/0TCRmi cells); (d) characterized by TCR gene
rearrangement; (e) precursor thymocytes that are CD4+CD8+ double positive (DP); (f) CD4-CD8+ orCD4+CD8- and optionally TCRh; (g) CD3+CD90+; (h) single positive (SP) cells that are CD4-CD8+ or CD4+CD8- and TCR'1'; (i) TCR-ab* and/or TCR-y6+; (j) characterized by expression of any of multiple nb chains (e.g. nb-3, -6, and 17a); or (k) mature and functional or activated T cells which may be characterized as TCR/CD3h', CD4-CD8+ or CD4+CD8-.
[0080] In one embodiment, a cell of the T cell lineage is a “progenitor T cell’ or
“proT cell”. The term “progenitor T cell” or “proT cell” as used herein means a T cell that is capable of maturing into a mature T cell or lymphocyte. In one embodiment, the pro T cell is a CD45+CD34+CD7+ proT cell.
[0081] In another embodiment, the progenitor T cell is a human progenitor T cell.
Phenotypes of human progenitor T cells include CD34+CD7+ and CD7+CD5+CD1a-.
[0082] The present inventors showed that human RAG2-deficient developing T cells progress up to the CD4+CD8+ double positive stage. Accordingly, in another embodiment, a cell of the T cell lineage is a CD4 and CD8 double positive (DP) cell characterized by a CD4+CD8+ or CD4+CD8+CD3+ phenotype.
[0083] In another embodiment, a cell of the T cell lineage is a CD4 or CD8 single positive (SP) cell characterized by a CD4-CD8+, CD4+CD8- or CD4-CD8+CD3+, CD4+CD8-CD3+ phenotype.
[0084] As used herein an “unrearranged T-cell receptor (TCR) gene locus” refers to a TCR gene locus which has not undergone rearrangement and remains in the germline configuration. Similarly, the term “unrearranged B-cell receptor (BCR) gene locus” refers to a BCR gene locus which has not undergone rearrangement and remains in the germline configuration.
[0085] The TCR gene locus can comprise an alpha (a) chain and a beta (b) chain (encoded for example by genes TRA and TRB, respectively), or a gamma and delta (g/d) chains (encoded for example by genes TRG and TRD, respectively). As will be understood by a person of skill in the art, during development of a T cell, rearrangement of segments of the genes encoding the TCR gene locus occurs to encode for antigen-specific receptors.
[0086] As used herein, the term “gene or protein required for V(D)J recombination” refers to a gene of protein that is required for correct rearrangement of the B- or T-cell receptor (BCR or TCR) gene loci. In one embodiment, the gene or protein
required for V(D)J recombination is a lymphocyte specific gene such as RAG1 or RAG2 or TdT.
[0087] In another embodiment, the gene or protein required for V(D)J recombination is a member of the non-homologous end joining (NHEJ) pathway for DNA repair.
[0088] In another embodiment, the gene or protein required for V(D)J recombination is Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair crosscomplementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1 ; also known as Cernunnos or XRCC4-like factor (XLF)), Paralog of XRCC4 and XLF (PAXX), or the DNA polymerases l and m.
[0089] As noted above, in one embodiment, the gene or protein required for
V(D)J recombination is RAG1 or RAG2. The V(D)J Recombination Activating Genes (RAG) 1 and 2 encode two essential DNA processing enzymes required for the rearrangement of the B- and T-cell receptor (BCR orTCR) gene loci (Schatz at al, 1989; Oettinger et al, 1990). As described above, the inventors differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2- KO) and showed that human RAG2-deficient developing T-cells progress up to the CD4+CD8+ stage.
[0090] Accordingly, in the methods disclosed herein, a cell of the T cell lineage or B cell lineage may be generated by culturing a sample comprising stem cells or progenitor cells, wherein the expression and/or function of RAG1 and/or RAG2 in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells.
[0091] As used herein, RAG1 refers to V(D)J Recombination Activating Protein
(RAG) 1 , which is encoded by the RAG1 gene. The term “RAG1” includes RAG1 from any species or source. The term also includes sequences that have been modified from any of the known published sequences of RAG1 proteins and genes. RAG1 orthe RAG1 gene may have any of the known published sequences for RAG1 which can be obtained from public sources such as GenBank. In one embodiment, RAG1 is human RAG1. Examples of human amino acid sequences for RAG1 include GenBank accession no. AAQ13571.1. Examples of human nucleic acid sequences for RAG1 include include GenBank no. NM_001377277.1 (Gene ID: 5896). The aforementioned sequences are incorporated herein by reference.
[0092] RAG2 refers to V(D)J Recombination Activating Protein (RAG) 2, which is encoded by the RAG2 gene. The term “RAG2” includes RAG2 from any species or source. The term also includes sequences that have been modified from any of the known published sequences of RAG2 proteins and RAG2 genes. RAG2 or the RAG2 gene may have any of the known published sequences for RAG2 which can be obtained from public sources such as GenBank. In one embodiment, RAG2 is human RAG2. Examples of the human amino acid sequences for RAG2 include GenBank accession no. AAH22397.1. Examples of human nucleic acid sequences for RAG2 include GenBank accession no. NMJD00536.4 (Gene ID: 5897). The aforementioned sequences are incorporated herein by reference.
[0093] As used herein, the term “RAG1 expression” includes both RAG1 protein expression and RAG1 gene expression. Likewise, term “RAG2 expression” includes both RAG2 protein expression and RAG2 gene expression.
[0094] As used herein, the term “RAG1 function” and “RAG2 function” refers to the biological activity of the RAG1 and RAG2, respectively. For example, cells lacking the biological activity of RAG1 and/or RAG2 enzymes are unable undergo rearrangement of the B cell receptor and T cell receptor (BCR or TCR) gene loci.
[0095] Endogenous expression and/or function of a gene or protein required for
V(D)J recombination (for example endogenous expression and/or function of RAG1 and/or RAG2) can be reduced or eliminated using any of a number of methods known in the art.
[0096] Reduced or eliminated expression and/or function of a gene or protein required for V(D)J recombination may be accomplished by mutation of deletion of the corresponding gene sequence. Reduced or eliminated expression and/or function of RAG1 or RAG2 may be accomplished for example by mutation or deletion of RAG1 or RAG2 gene sequences. For example, RAG1 or RAG2 gene sequences may be mutated or deleted from the genome of pluripotent stem cells using, for example, gene editing methods. Thus, for example, approaches employing RNA/DNA guided endonucleases (e.g., Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9, Cpf1 , and Argonaute), Transcription Activator- Like Effector (TALE)-nucleases, zinc finger nucleases (ZFN), or meganucleases can be adapted for use in embodiments of the present disclosure. In various examples, insertions or deletions are made by gene editing to cause a frame shift mutation, leading to gene knock out (i.e., lack of expression of a functional gene product).
[0097] Specific examples of protocols used in the present invention in the context of RAG2 include transfection of an expression vector containing cassettes for GFP, Cas9 endonuclease, a CRISPR chimeric cDNA and a gRNA moiety that targets RAG 2.
[0098] In another embodiment, a RAG1 and/or RAG2 inhibitor is used to reduce endogenous RAG1 and/or RAG2 expression and/or function. The term “inhibitor” refers to an agent that reduces, decreases or otherwise blocks expression or activity of its target and includes any substance that is capable of inhibiting the expression or activity of the target and includes, without limitation, small molecules, antisense oligonucleotide molecules (antisense nucleic acid molecules), siRNAs or shRNAs, aptamers, proteins, antibodies (and fragments thereof), gene editing agents and other substances directed at the target expression or activity.
[0099] Endogenous RAG1 and/or RAG2 expression and/or function can be reduced transiently or permanently (for example, by introducing gene mutations or deletions into the germline).
[00100] As used herein, the term “wildtype” refers to a cell which has normal (non- modified), endogenous expression levels of a gene or protein required for V(D)J recombination. In one embodiment, the term “wildtype” refers to a cell which has normal (non-modified), endogenous expression levels of RAG1 and/or RAG2 genes or proteins. The wildtype cell is optionally a stem or progenitor cell.
[00101] In one embodiment of the present disclosure, endogenous RAG1 expression and/or function is reduced by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG1 expression and/or function.
[00102] In another embodiment, endogenous RAG2 expression and/or function is reduced by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG2 expression and/or function.
[00103] In one embodiment, the stem or progenitor cell is a pluripotent stem cell. As used herein, the term "pluripotent stem cell" refers to any stem cell having the potential to differentiate into all cell types of a human or animal body, not including extra- embryonic tissues. These stem cells include both embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). Hence, the cells suitable for the method of the present
invention include stem cells selected from iPSCs and ESCs. In one embodiment, the pluripotent stem cells are human pluripotent stem cells (hPSCs) and they include human iPSCs (hiPSCs) and human ESCs (hESCs).
[00104] The term “embryonic stem cell” or “ESC” as used herein refers to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germ line of a developing embryo.
[00105] The term “induced pluripotent stem cell” or “iPSC” as used herein refers to cells derived from somatic cells, such as skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state. In one embodiment, iPSCs are derived from T cells with a known or unknown TCR specificity (for example, T cells bearing TCRs with specificity against cancer).
[00106] In one embodiment, the stem or progenitor cell is a hematopoietic stem or progenitor cell (HSPC). In another embodiment, the stem or progenitor cell is a CD34+ hematopoietic precursor cell, optionally a CD34+ hemogenic endothelial precursor cell that has been differentiated from an ESC or iPSC, or a CD34+ pre-hematopoietic cell differentiated from an ESC or pluripotent stem cell (PSC). Various differentiation protocols for obtaining CD34+ cells are known in the art. For therapeutic applications, the stem cells or progenitor cells used to generate the cells of the T cell lineage may be obtained from the patient to be treated.
[00107] Stem or progenitor cells may be obtained from any suitable source, including, without limitation, umbilical cord blood, embryos, embryonic tissue, fetal tissue, bone marrow and blood.
[00108] Typically, a sample containing stem or progenitor cells is first depleted of non-stem cells or mature cells. Negative and positive selection methods known in the art may be used for enrichment of the stem or progenitor cells. For example, cells can be sorted based on cell surface antigens using a fluorescence activated cell sorter, or magnetic beads which bind cells with certain cell surface antigens. Negative selection columns can be used to remove cells expressing lineage specific surface antigens.
[00109] In an embodiment, a sample containing stem or progenitor cells is separated into lineage-negative (Lin ) and lineage position (Lin+) fractions. The Lin- fraction can be sorted for CD34+ cells.
[00110] The progenitor cells or stem cells may be cultured under suitable conditions to generate cells of the T cell lineage or B cell lineage. Methods of culturing
progenitor cells or stem cells to generate cells of the T cell lineage or B cell lineage are known in the art.
[00111] For example, as described herein, human pluripotent stem cells may be induced to differentiate as embryoid bodies, then CD34+ cells may be isolated by magnetic-assisted cell-sorting and placed in co-culture with OP9-DL4 cells to induce their differentiation towards the T cell. Such a protocol is described in Kennedy et al. 2012.
[00112] In one embodiment, the cells are cultured in the presence of one or more Notch ligands conjugated to a suspension support for a sufficient time to form cells of the T cell lineage.
[00113] The progenitor cells or stem cells may be cultured on plates or in suspension in a bioreactor, optionally a closed or a closed, automated bioreactor. Various bioreactors are known in the art and can include batch, fed batch or continuous bioreactors. An example of a continuous bioreactor is a continuous stirred-tank reactor model.
[00114] Various concentrations of progenitor cells or stem cells in the culture are contemplated. For example, the concentration of progenitor cells or stem cells in the culture may be anywhere from 1 to millions of cells per ml of media.
[00115] One or more positive cytokines that promote commitment and differentiation of cells of the T cell lineage or B cell lineage may also be added to the culture. The cytokines may be human in origin, or may be derived from other species. The concentration of a cytokine in a culture is typically about 1-10ng/ml. The following are representative examples of cytokines that may be employed in the present application to promote commitment and differentiation of cells of the T cell lineage: all members of the Flt-3-ligand, and interleukin-7 (IL-7) and Stem Cell Factor. In one embodiment, the cytokines used herein are Flt-3-ligand and IL-7 and Stem Cell Factor. The cytokines may be used in combination with equal molar or greater amounts of a glycosaminoglycan such as heparin sulfate. The cytokines are commercially available or can be produced by recombinant DNA techniques and purified to various degrees. Some of the cytokines may be purified from culture media of cell lines by standard biochemical techniques.
[00116] One or more additional molecules, optionally conjugated to a suspension support, may also be added to the culture. In one embodiment, the additional molecule
is a molecule that promotes T cell development (for example, promotes commitment and differentiation of cells of T cell lineage), also referred to herein as a “T cell co stimulatory molecule”. In one example, the inventors have shown that microbead- conjugated DL4 and VCAM1 cultured with HSPCs accelerated differentiation to the T cell lineage. Thus, in one embodiment, the T cell co-stimulatory molecule is VCAM 1. As used herein, the term “VCAM1” refers to Vascular cell adhesion protein 1 also known as vascular cell adhesion molecule 1 (VCAM1) or cluster of differentiation 106 (CD106), a protein that in humans is encoded by the VCAM1 gene. The term "VCAM 1 " also includes a mutant or variant of a VCAM1. In another embodiment, the T cell co-stimulatory molecule is a cytokine or chemokine (Stem Cell Factor, IL-7, CCL25, or CXCR4), Major Histocompatibility Complex (MHC) class I or class II, or co-stimulatory (CD80, CD86) molecule. Optionally, the T cell co-stimulatory molecule comprises at least one protein tag. Various protein tags are known in the art and can be used for a number of purposes. In one embodiment, the T cell co-stimulatory molecule comprises an Fc tag (also known as an Fc-fusion protein).
[00117] The progenitor cells and stem cells may be cultured in culture media comprising conditioned media, non-conditioned media, or embryonic stem cell media. Examples of suitable conditioned media include IMDM, DMEM, or aMEM, conditioned with embryonic fibroblast cells (e.g. human embryonic fibroblast cells or mouse embryonic fibroblast cells), or equivalent media. Examples of suitable non-conditioned media include Iscove’s Modified Dulbecco’s Medium (IMDM), DMEM, or aMEM, or equivalent media. The culture media may comprise serum (e.g. bovine serum, fetal bovine serum, calf bovine serum, horse serum, human serum, or an artificial serum substitute) or it may be serum free.
[00118] In one embodiment, the culture conditions entail culturing the progenitor cells or stem cells for a sufficient period of time so that cells in the preparation form proT cells. In another embodiment, the culture conditions entail culturing the progenitor cells or stem cells for a sufficient period of time so that cells in the preparation form mature T cells, for example mature SP T cells. It will be appreciated that the cells may be maintained for the appropriate amount of time required to achieve the desired cellular composition. Optionally, the progenitor cells or stem cells are cultured for at least 6, 8, 10, 12, 14, 21 , 28, 35 or 42 days.
[00119] In one embodiment, the method further comprises engineering the cells to comprise a nucleic acid encoding a T cell receptor (TCR) or a chimeric antigen
receptor (CAR). In another embodiment, the method further comprises engineering the cells to comprise a nucleic acid encoding a T cell receptor beta chain (TCRp).
[00120] The cells may be engineered to comprise a nucleic acid encoding a TCR, CAR and/or TCRp at any time during the differentiation process. For example, in one embodiment, the stem or progenitor cells comprise a nucleic acid encoding a TCR, CAR and/or TCRp. In another embodiment, the cells of the T cell lineage comprise a nucleic acid encoding a TCR, CAR and/or TCRp. In further embodiments, the stem or progenitor cells and/or the cells of the T cell lineage express a TCR, CAR and/or TCRp.
[00121] In one embodiment, the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp alone. In other words, the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and do not comprise a nucleic acid encoding a TCR or CAR.
[00122] In another embodiment, the stem or progenitor cells or the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and a nucleic acid encoding a CAR. In such an embodiment, the TCRp directs differentiation while the CAR can provide therapeutic efficacy.
[00123] In another embodiment, the method further comprises engineering the cells of the B-cell lineage to express a B cell receptor (BCR), B cell receptor beta chain (BCRp) and/or CAR. The cells may be engineered to comprise a nucleic acid encoding a BCR, BCRp and/or CAR at any time during the differentiation process. For example, in one embodiment, the stem or progenitor cells comprise a nucleic acid encoding a BCR, BCRp and/or CAR. In another embodiment, the cells of the B cell lineage comprise a nucleic acid encoding a BCR, BCRp and/or CAR. In further embodiments, the stem or progenitor cells and/or the cells of the B cell lineage express a BCR, BCRp and/or CAR.
[00124] The cells may be engineered by any method known in the art to comprise a nucleic acid encoding a TCR, TCRp, CAR, BCR or BCRp. For example, the cells may be transformed with viral or non-viral vectors carrying a TCR, TCRp, CAR, BCR or BCRp. Examples of viral vectors include, but are not limited to, retroviruses (including lentivirus), adenoviruses and adeno-associated viruses. Examples of non-viral vectors include, but are not limited to, nude DNA, minicircle DNA vectors, liposomes, polymerizers and molecular conjugates.
[00125] The TCR, BCR or CAR optionally confers specificity to an antigen such as a tumour associated antigen, a viral antigen or a self-antigen.
[00126] A “tumor associated antigen” is an antigen produced by tumor cells. Examples of tumor associated antigens include, but are not limited to, alphafetoprotein (AFP), arcinoembryonic antigen (CEA), CA-125, MUC-1 , Epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), WT1 and NYES01.
[00127] A “viral antigen” is an antigen encoded by a viral genome. Examples of viral antigens include, but are not limited to EBV, CMV, HIV and SARS viral antigens.
[00128] A “self-antigen” is an antigen that is produced by the subject. A selfantigen can be a tumor associated antigens. A T cell expressing a TCR or CAR conferring specificity to a self-antigen could be used for example to make regulatory T cells that block self-reactive T cells.
II. Pluripotent stem cells
[00129] As described herein, the present inventors generated RAG2-deficient (RAG2-KO) human pluripotent stem cell (hPSC) lines.
[00130] Accordingly, the disclosure also provides an isolated stem or progenitor cell, wherein expression of at least one gene or protein required forV(D)J recombination, optionally RAG 1 and/or RAG2, in the stem cell or progenitor cell is reduced or eliminated compared to a wildtype stem cell or progenitor cell. Optionally, the stem cell or progenitor cell is human cell.
[00131] In one embodiment, the stem cell is a pluripotent stem cell.
[00132] In another embodiment, the pluripotent stem cell is an embryonic stem cell or induced pluripotent stem cell (iPSC).
[00133] Reduced or eliminated expression and/or function of RAG 1 or RAG2 may be accomplished by any one of a number of methods known in the art.
[00134] In one embodiment, the stem cell or progenitor cell comprises a mutated or deleted RAG1 and/or RAG2 gene sequence. For example, insertions or deletions in the RAG1 and/or RAG2 gene sequences may cause a frame shift mutation, leading to gene knock out (i.e., lack of expression of a functional gene product).
[00135] In one embodiment of the present disclosure, endogenous RAG1 expression and/or function is reduced in the stem cell or progenitor cell by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG1 expression and/or function.
[00136] In another embodiment, endogenous RAG2 expression and/or function is reduced in the stem cell or progenitor cell by at least 5%, 10%, 25%, 50%, 75% or 100% compared to a wild-type cell. In another embodiment, the cell has no detectable endogenous RAG2 expression and/or function.
[00137] In addition, in one embodiment, the stem cell or progenitor cell further comprises a nucleic acid encoding a TCR, CAR, TCRp, BCR or BCRp. In another embodiment, stem cell or progenitor cell expresses a TCR, CAR, TCRp, BCR or BCRp.
[00138] In a further embodiment, the stem or progenitor cell comprises a nucleic acid encoding a TCRp and does not comprise a nucleic acid encoding a TCR or CAR.
IV. Cells of the T cell or B cell lineage
[00139] The disclosure further provides cells of the T cell lineage or B cell lineage generated by the methods, systems and kits described herein, or mitotic or differentiated cells that are progeny of the cells.
[00140] In one embodiment, the disclosure provides a “progenitor T cell’ or “proT cell” generated by the methods described herein. In another embodiment, the progenitor T cell is a human progenitor T cell, for example a human progenitor T cell characterized by CD34+CD7+, CD7+CD5+CD1a- or CD45+CD34+CD7+.
[00141] The disclosure also provides a double positive (DP) T-cell characterized by CD4+CD8+ or CD4+CD8+CD3+. The disclosure further provides a cell of the T-cell lineage that is a single positive (SP) cell characterized by CD4-CD8+, CD4+CD8- or CD8+CD3+, CD4+CD3+.
[00142] In one embodiment, the cells of the T cell lineage further comprise a nucleic acid encoding a TCR, CAR or TCRp. In another embodiment, the cells of the T cell lineage express a TCR, CAR or TCRp.
[00143] In a further embodiment, the cells of the T cell lineage comprise a nucleic acid encoding a TCRp and does not comprise a nucleic acid encoding a TCR or CAR.
[00144] In another embodiment, a cell of the T cell lineage generated by the methods described herein (for example, a progenitor T cell or a mature T cell) is engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[00145] The antigen is optionally a tumor associated antigen (TAA), viral antigen or self-antigen. T-cells engineered to express a TCR or a CAR to confer specificity to a TAA can be useful for treating conditions such as cancer.
[00146] Likewise, in another embodiment, the cells of the B cell lineage further comprise a nucleic acid encoding a CAR, TCRp, BCR or BCRp. In another embodiment, the cells of the B cell lineage express a CAR, BCR or BCRp.
[00147] In another embodiment, a cell of the B cell lineage generated by the methods described herein is engineered with a B cell receptor (BCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[00148] The antigen is optionally a tumor associated antigen (TAA), viral antigen or self-antigen. T-cells engineered to express a BCR or a CAR to confer specificity to a TAA can be useful for treating conditions such as cancer.
[00149] In another aspect, the present disclosure provides a pharmaceutical composition comprising isolated stem or progenitor cells or cells of the T cell lineage or B cell lineage generated by the methods described herein and a pharmaceutically acceptable diluent or carrier.
[00150] Suitable diluents and carriers are described, for example, in Remington's Pharmaceutical Sciences. On this basis, the compositions include, albeit not exclusively, solutions of the proT cells in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso- osmotic with physiological fluids.
[00151] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween™), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.
[00152] Pharmaceutical compositions also include cyropreservative solutions. In one embodiment, cells of the T cell lineage generated by the methods described herein are cryopreserved in appropriate media, for example pharmaceutically acceptable or GMP-grade media and optionally formulated for administration to a subject in need thereof.
[00153] Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
[00154] The compositions can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration. For parenteral administration, solutions of the pro-T cells described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.
[00155] Preferably the cells of the T cell lineage or B cell lineage are present in an amount effective for treating a disease state in a subject need thereof. In one embodiment the cell of the T cell lineage is present in an amount effective to enhance hematopoietic progenitor cell engraftment in a subject in need thereof. Optionally, the composition further comprises cells of the T cell lineage, or tissue for transplantation. In one embodiment the tissue comprises a thymus. In another embodiment the tissue comprises an organ.
III. Kits
[00156] Stem or progenitor cell as described herein may be prepared and packaged in kits for use in generating cells of the T cell lineage or cells of the B cell lineage.
[00157] Accordingly, also provided herein is a kit for producing cells of the T cell lineage or cells of the B cell lineage comprising a stem or progenitor cell, wherein the expression of RAG1 and/or RAG2 in the stem cell or progenitor cell is reduced or eliminated compared to wildtype stem cell or progenitor cells.
[00158] In one embodiment, the kit further comprises culture media for culturing a sample comprising stem cells or progenitor cells for producing cells of the T cell lineage or cells of the B cell lineage. Examples of culture media include conditioned media, non-conditioned media, or embryonic stem cell media. The culture media may comprise serum (e.g. bovine serum, fetal bovine serum, calf bovine serum, horse serum, human serum, or an artificial serum substitute) or it may be serum free.
[00159] In another embodiment, the kit further comprises one or more additional molecules. In one embodiment, the additional molecule is a molecule that promotes T cell development (for example, promotes commitment and differentiation of cells of T cell lineage), also referred to herein as a “T cell co-stimulatory molecule”. In another embodiment, the T cell co-stimulatory molecule is VCAM1.
[00160] The media optionally includes one or more cytokines that promote commitment and differentiation of cells of the T cell lineage or cells of the B cell lineage. The cytokines may be human in origin, or may be derived from other species. The concentration of a cytokine in a culture is typically about 1-10ng/ml. The following are representative examples of cytokines that may be employed in the present application: all members of the Flt-3-ligand, and interleukin-7 (IL-7) and Stem Cell Factor. In one embodiment, the cytokines used herein are Flt-3-ligand and IL-7 and Stem Cell Factor. The cytokines may be used in combination with equal molar or greater amounts of a glycosaminoglycan such as heparin sulfate. The cytokines are commercially available or can be produced by recombinant DNA techniques and purified to various degrees. Some of the cytokines may be purified from culture media of cell lines by standard biochemical techniques.
[00161] In one embodiment, the kit comprises one or more containers for the within-described reagents.
[00162] Printed instructions providing guidance in the use of the reagent(s) may also be included in the kit, in various embodiments. The term “instructions” or “instructions for use” typically includes a tangible expression describing the cells, culturing time periods, temperature, media conditions, and the like. For example, in one embodiment, the instructions describe a method comprising culturing a sample comprising stem cells or progenitor cells.
V. Therapeutic Applications
[00163] T cells and B cells engineered to recognize specific antigens have wide ranging therapeutic applications.
[00164] Accordingly, the disclosure provides method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells or progenitor cells to a subject in need thereof,
[00165] wherein the stem cells or progenitor cells are engineered to comprise at least one of a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a B cell receptor (BCR) that confers specificity to an antigen.
[00166] The disclosure also provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, wherein the expression of RAG1 and/or RAG2 in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the T cell lineage to a subject in need thereof, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) to confer specificity to an antigen.
[00167] The disclosure also provides a use of cells of the T cell lineage, generated by the methods described herein for treating a disease or condition in a
subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confer specificity to an antigen. The disclosure further provides a use of cells of the T cell lineage, generated by the methods described herein for preparation of a medicament for treating a disease or condition in a subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[00168] The disclosure also provides cells of the T cell lineage, generated by the methods described herein for use in treating a disease or condition in a subject, wherein the cells of the T cell lineage are engineered with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers specificity to an antigen.
[00169] The disclosure also provides a method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the B cell lineage, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the B cell lineage to a subject in need thereof,
[00170] wherein the cells of the B cell lineage are engineered with at least one B cell receptor (TCR) to confer specificity to an antigen.
[00171] The disclosure also provides a use of cells of the B cell lineage, generated by the methods described herein for treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen. The disclosure further provides a use of cells of the B cell lineage, generated by the methods described herein for preparation of a medicament for treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen.
[00172] The disclosure also provides cells of the B cell lineage, generated by the methods described herein for use in treating a disease or condition in a subject, wherein the cells of the B cell lineage are engineered with a B cell receptor (BCR) or CAR that confers specificity to an antigen.
[00173] The antigen is optionally a tumor associated antigen (TAA), a viral antigen or a self-antigen. In one embodiment, the antigen is a TAA and the disease is cancer.
[00174] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex, weight of the subject. The amount of a given cell preparation that will correspond to such an amount will vary depending upon various factors. Such as the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. An “effective amount” will preferably be an amount effective for the cell of the T cell lineage to engraft the subject being treated.
[00175] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also includes prophylactic treatment.
[00176] The term “subject” as used herein means any member of the animal kingdom and is preferably a human.
[00177] The following non-limiting examples are illustrative of the present application:
EXAMPLES
Example 1
Materials and Methods:
[00178] hESC Maintenance. Human ESCs (H1 ; WiCell Research Institute, Madison, Wl) were maintained and expanded on plates coated with Matrigel (Corning,
NY, USA) in TeSR-E8 medium (STEMCELL Technologies, Vancouver, Canada). Cells were passaged by non-enzymatic dissociation using 0.5 mM EDTA.
[00179] Generation of RAG2-KO hESCs. The pD1321-GFP expression vector, containing cassettes for GFP, Cas9 endonuclease, a CRISPR chimeric cDNA, and the gRNA moiety designed to target RAG 2 [G GTT AT G CTTT ACAT CCAG A (SEQ ID NO: 1)], was custom synthesized (DNA2.0). After transfection with Lipofectamine 3000 (Life Technologies, Carlsbad, CA), GFP+ hESCs were sorted using flow cytometry. Individual clones were picked, expanded, and aliquots were collected for purification of genomic DNA using the Genomic DNA kit (Invitrogen). Mutations (indels) were validated by sequencing products of PCR amplification of the regions flanking the targeting sites. The RAG2 KO clone 1 exhibited a 1 bp deletion in one allele and 16bp deletion in the other allele, while clone 4 exhibited a 11 bp deletion in one allele and 23bp deletion in the other allele.
[00180] Western Blot Analysis. Briefly, cellular lysates were prepared by incubating the cells in lysis buffer (50 mM Tris-HCI, pH7.5, 150 mM NaCI, 0.5% NP-40, 2 mM EDTA) containing protease inhibitor cocktail (Roche) for 20 min at 4°C, followed by centrifugation at 14000*g for 15 min at 4°C. Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore, Louis, MO) and probed with anti-RAG2 antibody (Abeam - Ab95955; 1 :1000 dilution) overnight at 4°C followed by incubation with secondary antibody. The anti-RAG2 antibody used was a rabbit polyclonal made against a recombinant fragment corresponding to amino acids 271-519 of human RAG2 (Abeam), which are well beyond the gRNA targeting site. Immunoreactive bands were visualized using western blotting Luminol reagent (Thermo). PBMCs from a T-ALL patient were used as positive control (Bories et al, 1991).
[00181] Immunostaining. Cells were fixed in 4% paraformaldehyde, and permeabilization and blocking were performed in 5% NGS (Abeam, Cambridge, USA) and 1% Triton X-100 (Sigma-Aldrich, Louis, MO) in PBS for 30 min. Cells were stained and analyzed as previously described (Li et al, 2017).
[00182] Teratoma Formation. All animal studies were approved by Ethics Committee of Experimental Research of Peking University and were in accordance with the International Animal Care and Use Committee Guidelines. 6-8 week-old non-obese diabetic (NOD)/SCID mice were injected subcutaneously with 1*107 hESCs resuspended in DMED-F12 with 50% Matrigel to allow teratoma formation for 8 weeks. Histological analysis was performed as previously described (Li et al, 2017) .
[00183] hPSC Differentiation and CD34+ Isolation. hPSCs, H1 embryonic stem cells (ESC) were differentiated as previously described (Kennedy et al, 2012). After 8 days of differentiation, embryoid bodies were harvested and dissociated into single-cells using Collagenase type IV and trypsin-EDTA as previously described (Kennedy et al, 2012), and positively selected using a MACS column (Miltenyi Biotec) with anti-CD34 PE-conjugated antibody (BD Biosciences) and anti-PE microbeads. The cell yield and purity of the positive selection was assessed pre- and post- MACS by flow cytometry.
[00184] OP9-DL4-7FS Co-Culture and Differentiation. OP9-DL4 cells expressing hlL-7, hFLT3-L, and hSCF (7FS) were generated and grown in a-MEM containing 10-20% FBS (Gibco), 1% Pen-Strep (Life Technologies), and phospho- ascorbic acid (Sigma-Aldrich) at 37°C, 5% CO2. For co-cultures, OP9-DL4-7FS cells were plated in 6-well plates the previous day. MACS-purified CD34+ cells were counted and seeded at a density of 5-20 x104 cells per 6-well plate, and differentiated by coculture with OP9-DL4-7FS cells in OP9 medium at 37°C, 5% CO2, and cell were passaged and co-cultured with fresh OP9-DL4-7FS cells every ~5 days.
[00185] Retroviral Transduction. PG13 cell lines stably expressing empty vector dTomato, TCRa-dTomato, or TCRp-dTomato were grown towards 70% confluency, at which point the media was switched to a-MEM with 15% FBS and 1% Pen-Strep to condition the supernatant for 48 h before transducing OP9-DL4-7FS/T-cell cultures. On D8+24 of OP9-DL4-7FS/T-cell cultures, cells were transduced with corresponding PG13 supernatants once a day with 1 mI/ml polybrene and centrifuged at 2000xg for 90 min at RT for 4 d (2-3x10® cells per 2 ml of supe). Cells were rested for 3 d and then sorted as dTomato+ CD7+CD5+CD4+CD8+ DPs. Sorted cells were placed in OP9-DL4-7FS co-cultures for up to 10 days.
[00186] Flow Cytometry. Cells were stained for 30 min on ice with the following mouse anti-human antibodies: CD3-BrilliantViolet421 , CD4-AlexaFluor700, CD8P-PE, CD31-FITC, CD34-PE, CD45RA-PE/CF594, TCRgd-FITC (BD Biosciences), CD5- PE/Cy7, CD7-AlexaFluor700, CD45-APC/eFluor780, TCRab-APC (eBiosciences), CD8a-PE/Dazzle, CD38-BrilliantViolet421 (BioLegend). Cells were resuspended in flow cytometry buffer containing DAPI, data was collected using LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo version 9.7.6. For intracellular staining, cells were fixed and permeabilized using Fixation/Permeabilization kit with GolgiPlug™ (BD Biosciences) as per manufacturer’s instructions.
[00187] RNA-Seq Analysis of Control and RAG2-KO in vitro Derived CD4+CD8+ cells. Cells were collected at co-culture day 24-28 and stained with fluorochrome-labelled antibodies to CD45, CD7 (eBioscience), CD5, CD4, CD8 (BD Biosciences), DAPI, and sorted into CD4+CD8+ populations using FACSVantage Diva or FACSAria cell sorters (BD Biosciences). Total RNA was extracted from the sorted cell populations using TRIzol. Purified RNA was subjected to RNA sequencing using lllumina Novaseq 6000. Library preparation was done using lllumina TruSeq Strandard Total RNA Sample Preparation kit. Sequencing was done using 100-cycle paired read protocol and multiplexing to obtain ~40 million reads/sample. Samples were aligned to GRCh38 using HISAT2. Read counts were calculated using HTSeq. Differential gene expression analysis was done using edgeR package in the R platform.
[00188] Statistical Analysis. The data and error bars are presented as mean + standard error of mean. To determine statistical significance, a one-way ANOVA (comparing three means) was performed using Prism version 6. Statistical significance was determined as *P< 0.05 and **P< 0.01.
[00189] Data Availability. Raw and processed RNA-Seq data are available from the Gene Expression Omnibus under accession number GSE164276 (https://www.ncbi. nlm.nih.qov/qeo/querv/acc.cqi?acc=GSE164276').
Results and Discussion
[00190] Generation and characterization of RAG2-KO hPSC lines. To evaluate the role of RAG2 in human T-cell development, CRISPR-Cas9 gene editing was used to target exon 3 of the RAG2 gene (Fig. 1A). hPSCs were transfected with a plasmid encoding the RAG2-targeting guide RNA, the Cas9 enzyme, and green fluorescent protein (GFP). Transfected GFP+ hPSCs were single-cell sorted and cultured. After expanding individual clones, two clones were identified that contained unique insertion-deletions with bi-allelic mutations (KO-1 and KO-4) (Fig. 1A). To evaluate the impact of the RAG2 mutations on protein expression, Western blot analysis was performed, which confirmed the absence of detectable RAG2 protein in both KO-1 and -4 derived T-lineage cells (Fig. 1B).
[00191] To assess whether RAG2-KO hPSCs maintained pluripotency, the expression of key markers and teratoma formation was evaluated. Immunofluorescence staining showed that RAG2-KO hPSCs expressed OCT4, NANOG, SOX2, and SSEA- 4 (Fig. 1C). To functionally test pluripotency, RAG2-KO hPSCs were injected into immunodeficient mice, and histological analysis revealed RAG2-KO teratoma formation
with all three germ layers (Fig. 1D), indicating that RAG2-K0 hPSCs retained key features of pluripotency. To determine the capacity of RAG2-KO hESCs to generate hematopoietic progenitors, CD34 expression was analyzed after 8 days of embryoid- body differentiation cultures (Kennedy et al, 2012). Control WT, RAG2-KO-1 and -4 hPSCs gave rise to similar frequencies of hemogenic endothelial CD34+ cells, which could be further enriched by magnetic-assisted cell sorting (MACS) (Fig. 1E).
[00192] T-cell development from RAG2-KO hPSCs. CD34+ hemogenic endothelial cells were MACS-enriched and cultured with OP9-DL4 cells, expressing human IL-7, FLT3-ligand and stem cell factor (7FS), to induce T-cell differentiation. After 10 days of culture, Control WT, RAG2-KO-1 and -4 cells proceeded along the T-cell lineage, as marked by expression of both CD7 and CD5 (Fig. 2A). All three groups reached the CD4+ intermediate single positive (ISP) stage by day 15 and displayed intracellular CD3 expression by day 20 (Fig. 2A). After 24 days of culture, the majority of cells from Control WT, RAG2-KO-1 and -4 groups were CD7+CD5+ (Fig. 2A). Of note, by 29-34 days of culture, Control WT, RAG2-KO-1 and -4 cells all reached the CD4+CD8+ DP stage (Fig. 2A). However, as expected, only Control WT cells displayed intracellular TCRp expression, and cell surface CD3/TCRP expression (Fig. 2A). These results indicate that RAG2-deficient human T-cell progenitors can differentiate up to the CD4+CD8+ DP stage.
[00193] To determine cell survival and expansion, total cellularity was quantified from Control WT, RAG2-KO-1 and -4 developing T-cells. After 20 days of culture, all three samples showed similar cell numbers (Fig. 2B). However, after 40 days of culture, Control WT T-lineage cells further increased their survival and expansion as opposed to RAG2-KO-1 and -4 T-lineage cells, with approximately 10-fold greater cellularity by this time-point (Fig. 2B).
[00194] Forced expression of a TCRp chain in RAG2-KO CD4+CD8+ DPs.
RAG2-KO hPSC-derived CD34+ cells were cultured on OP9-DL4-7FS cells for 24 days, and DP cells were retrovirally-transduced with an empty vector (dTomato), a rearranged TCRa chain (TRA 1383Ϊ), or a rearranged TCRp chain (TRB 1383i) (Fig. 3A). Transduced RAG-KO cells were sorted for CD7+CD5+CD4+CD8+ DP cells, and placed back on OP9-DL4-7FS cells for an additional 10 days to assess for cell survival and expansion (Fig. 3A). Both dTomato- and TCRa-transduced DPs showed similar cell numbers after 10 days of culture (Fig. 3B). However, TCRp-transduced DPs displayed significantly higher cell numbers after 10 days of culture compared to dTomato- and
TCRa-transduced DPs (Fig. 3B). Without being bound by theory, this suggests that, in contrast to a TCRa chain, expression of a rearranged TCRp chain promotes cell survival and/or proliferation of developing human T-cells at the DP stage.
[00195] Transcriptomic analysis of Control WT, RAG2-KO, and RAG2-KO TCRp-transduced CD4+CD8+ DP cells. Control WT, RAG2-KO control-transduced, and RAG2-KO TCRp-transduced DP cells were sorted for RNA sequencing (RNA-Seq) analysis. Control WT DP cells expressed a large set of TCRa, TCRp, TCRy, and TCR6 genes that were absent in RAG2-KO control-transduced and RAG-KO TCRp- transduced DP cells, with the notable exception of some TCR genes, including the 1383i TCRp used in the transduction and a few TCRa genes, likely the result of germline transcripts induced by the b-selection signals, as seen in mice (Villey et al, 1997) (Table 1, and Fig. 4A). Furthermore, the expression of genes from RAG2-KO control- and TCRp-transduced DP cells were compared with that of umbilical cord-blood (UCB)- hematopoietic stem cell-derived DP cells for a set of DP-associated signature genes (Casero et al, 2015) (Table 1). This analysis revealed that TCRp-transduced RAG2-KO DP cells gained the expression of a suite of genes that were present in the UCB-derived DP cells, including RORC, which is induced following b-selection in mice (He 2000) (Fig. 4B).
[00196] Differentially expressed gene analysis was performed to determine genes that were up-regulated in Control WT compared to RAG2-KO (KO-1 and KO-4) DP cells, and vice versa. Genes significantly up-regulated in Control WT compared to both RAG 2- KO-1 and RAG2-KO-4 include CCDC152, GPR183, IL32 , and MAL (Table 1 and Fig. 5A). Genes significantly up-regulated (p<0.05) in Control WT compared to RAG 2- KO-1 or -4 included ADAMTS17, IL1RL1 , PLXNA2, and TEAD1, or CTSW , IKBKG, IL21R, and IL4R, respectively (Table 1 and Fig. 5A). Of note, many of these genes (such as TEAD1, IKBKG, and IL32) were also highly expressed in TORb- transduced RAG2-KO DP cells similar to Control WT DP cells (Fig. 5A). Interestingly, the expression of a subset of genes (such as MAL) were not rescued with forced expression of a TORb chain (Fig. 5A). Genes significantly up-regulated in both RAG2- KO-1 and RAG2-KO-4 compared to Control WT include HBG1, HBG2, LOC100240735, and LOC339975 (Table 1 and Fig. 5B). Genes significantly up-regulated in RAG2-KO- 1 or -4 compared to Control WT include CCDC8, CPA4, EPHA4, and ID1, or ANKRD1,
CMTM8, MET , and RHOU, respectively (Table 1 and Fig. 5B). Of note, many of these genes (such as ID1, RHOU, and HBG1) also showed low expression in TCRp- transduced RAG2-KO DPs similar to Control WT DP cells (Fig. 5B). Interestingly, the expression of a subset of genes (such as LOC100240735) were not reduced with forced expression of a TCRp chain (Fig. 5B). Phylogenetic tree analysis again showed that TCRp-transduced RAG2-KO DP cells were more similar to Control WT DP cells than to control-transduced RAG2-KO DP cells (Fig. 5).
[00197] Analysis of cell cycle regulators, survival and differentiation genes, which are known to be involved in mouse b-selection (Lefebvre et al, 2005; Klein et al, 2019; Sicinska et al, 2003), revealed a set of genes, such as RORC, CD27, ERG and CCDN3, that are also regulated following TCRp expression in RAG2-KO DPs (Fig. 6A). A gene ontology analysis to determine biological pathways that involve genes up-regulated in Control WT compared to RAG2-KO DP cells revealed a genetic program involved in “negative regulation of intrinsic apoptotic signaling pathway” (Fig. 6B). Furthermore, a gene set pathway pertaining to leukocyte regulation, included genes involved in “positive regulation of lymphocyte activation” and “regulation of lymphocyte proliferation” (Fig. 6B). Thus, these data provide additional evidence that RAG2-dependent TCRp expression in developing human T-cells supports their survival and/or proliferation.
Summary
[00198] RAG2-KO hPSCs were generated to assess the role of TCRp during human T-cell development. In contrast to the effects seen with Rag1- or Rag2-deficient mice, which show a complete lack of DP thymocytes, developing human T-cells were able to reach the CD4+CD8+ DP stage in the absence of RAG2 expression. Lack of RAG 1/2 in mice results in a definitive block at the CD44 CD25+ DN3 stage, as it is well documented that RAG1/2-mediated TCRp rearrangement controls the transition from the DN3 to the CD44 CD25- DN4 and DP stages (von Boehmer et al, 1999; Michie and Zuniga-Pflucker, 2002). However, the precise developmental block in the absence of RAG1/2 expression or TCRp rearrangement during human T-cell development was largely unresolved (Rothenberg and Taghon, 2005; Carrasco et al, 2002).
[00199] It was previously suggested that the requirement for TCRp-induced survival/proliferation, or b-selection, occurs at the CD4+ ISP stage, in which 5% of cells express cell-surface TORb protein (Blom et al, 1999). In another study, it was suggested
that b-selection instead happens later, at the CD4+CD8a+CD8p_ early double positive (EDP) stage in which 25% of cells express intracellular TCRp protein (Carrasco et al, 1999). Thus, based on these two studies, it was proposed that b-selection begins at the CD4+ ISP stage and continues until the EDP stage. Here, robust development was detected past the ISP and EDP to the DP stage, as CD4, CD8a, and Oϋdb expression was observed in RAG2-KO developing T-cells. While the requirement for the TCRb chain in the phenotypic differentiation to the DP stage differs between mice and humans, the TORb chain similarly promotes cellular expansion (survival/proliferation) in both mouse and human developing T-cells, as enforced TORb expression rescued cellular expansion. Without being bound by theory, the requirement for TORb, and thus the preTCR, during, rather than prior to, the DP stage during human T-cell development may reflect differential expression of cell cycle and survival genes. In mice, expression of a preTCR in DN3 cells leads to expression of Bmi-1 , which represses the expression of the cell cycle inhibitor Cdkn2a. Repression of Cdkn2a is required for preTCR-induced cell proliferation and the DN3-DP transition (Miyazaki et al, 2008). In summary, this study reveals the unexpected timing required for TCRb-mediated b-selection in developing human T-cells.
Example 2
Forced expression of a TCRc^ and TCRyS chains in RAG2-KO CD4+CD8+ DPs [00200] RAG2-KO hPSC-derived CD34+ cells were cultured on OP9-DL4-7FS cells for 21 days, and DP cells were retrovirally-transduced with an empty vector (dTomato), rearranged TCRa^ chains (1383i-TCR) (Roszkowski et al., 2003), or rearranged TCRy8 chains (3C2-TCR) (Benveniste et al., 2018) (Fig. 7). Transduced RAG-KO cells were sorted for CD7+CD5+CD4+CD8+ DP cells, and placed back on OP9- DL4-7FS cells for an additional 4 and 10 days to assess for cell survival and expansion (Fig. 7A). TCRa^- and TCRy8-transduced DPs displayed higher cell numbers after 4 and 10 days of culture compared to dTomato-transduced DPs (Fig. 7A), with TORab- transduced cells showing a much higher fold expansion than TCRy5-transduced cells, and akin to what was seen with TCRb-transduced DP cells (Fig. 3). Without being bound by theory, this suggests that similar to a TORb chain alone (preTCR, pTa/TCRb), expression of a rearranged TCRa^ or TCRy5 chains promote cell survival and/or proliferation of developing human T-cells at the DP stage. Additionally, flow cytometric
analysis of TCR-transduced RAG2-KO DP cells showed the expression of the corresponding ab and gd TCRs, respectively, on the cell surface of T-lineage cells (Fig. 7B). These results show that developing T cells obtained from RAG2-deficient PSCs differentiated in vitro can be transduced to effectively express a gd or ab TCR.
Example 3
[00201] CRISPR-Cas9 gene editing is used to target each of the following genes: Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1 ; also known as Cernunnos orXRCC4-like factor (XLF)), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
[00202] hPSCs are transfected with a plasmid encoding the gene targeting guide RNA, the Cas9 enzyme, and green fluorescent protein (GFP). Transfected GFP+ hPSCs are single-cell sorted and cultured.
[00203] To assess whether various KO hPSCs maintained pluripotency, the expression of key markers and teratoma formation are evaluated. Immunofluorescence staining shows that the KO hPSCs express OCT4, NANOG, SOX2, and SSEA-4.
[00204] To functionally test pluripotency, the KO hPSCs are injected into immunodeficient mice, and histological analysis reveals KO teratoma formation with all three germ layers, indicating that KO hPSCs retain key features of pluripotency. To determine the capacity of KO hESCs to generate hematopoietic progenitors, CD34 expression is analyzed after 8 days of embryoid-body differentiation cultures (Kennedy et al, 2012). Control WT and KO hPSCs give rise to similar frequencies of hemogenic endothelial CD34+ cells.
[00205] T-cell development from KO hPSCs. CD34+ hemogenic endothelial cells are MACS-enriched and cultured with OP9-DL4 cells, expressing human IL-7, FLT3-ligand and stem cell factor (7FS), to induce T-cell differentiation. After 10 days of culture, Control WT and KO cells proceed along the T-cell lineage, as marked by expression of both CD7 and CD5. All three groups reach the CD4+ intermediate single positive (ISP) stage by day 15 and display intracellular CD3 expression by day 20. After 24 days of culture, the majority of cells from Control WT and KO groups are CD7+CD5+ By 29-34 days of culture, Control WT and KO cells all reach the CD4+CD8+ DP stage.
[00206] To determine cell survival and expansion, total cellularity is quantified from Control WT, and KO developing T-cells. After 20 days of culture, all three samples show similar cell numbers.
[00207] Forced expression of a TCRp chain in KO CD4+CD8+ DPs. KO hPSC- derived CD34+ cells are cultured on OP9-DL4-7FS cells for 24 days, and DP cells are retrovirally-transduced with an empty vector (dTomato), a rearranged TCRa chain (TRA 1383i), or a rearranged TCRp chain (TRB 1383i). Transduced KO cells are sorted for CD7+CD5+CD4+CD8+ DP cells, and placed back on OP9-DL4-7FS cells for an additional 10 days to assess for cell survival and expansion. Both dTomato- and TCRa-transduced DPs show similar cell numbers after 10 days of culture. However, TCRp-transduced DPs display significantly higher cell numbers after 10 days of culture compared to dTomato- and TCRa-transduced DPs.
Example 4
Generation of KO SP T cells Generation of KO SP T cells. KO hPSCs engineered with one or more of a rearranged TCRpchain (TRB 1383i), a CAR or a TCR are differentiated into CD34+ cells and subsequently CD4+CD8+ DP cells as above or in stromal cell-free conditions (see for example WO2019157597A1 , the contents of which are incorporated by reference in their entirety). Generated CD4+CD8+ DP cells are further differentiated into CD4-CD8+ SP and/or CD8-CD4+ SP T cells. KO DP cells engineered with TCRp successfully progress to SP T cells in comparison with KO-only cells.
Table 1. List of genes significantly up-regulated (p<0.05) in Control WT DPs compared to RAG2-KO-1 DPs, or vice versa, as indicated: (NA: non-assigned TCR gene names).
Table 2. List of genes significantly up-regulated (p<0.05) in Control WT DPs compared to RAG2-KO-4 DPs, or vice versa, as indicated: (NA: non-assigned TCR gene names).
Table 3. List of genes significantly up-regulated (p<0.05) in thymus DNs (CD34+CD7+CD1a+CD4-CD8-) compared to thymus DPs (CD4+CD8+), or vice versa from Casero et al., 2015 (18), as indicated: (NA: non-assigned TCR gene names).
REFERENCES
Schatz, D. G., M. A. Oettinger, and D. Baltimore. 1989. The V(D)J recombination activating gene, RAG-1. Cell 59: 1035-1048.
Oettinger, M. A., D. G. Schatz, C. Gorka, and D. Baltimore. 1990. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248: 1517- 1523.
Jones, J. M., and M. Gellert. 2004. The taming of a transposon: V(D)J recombination and the immune system. Immunol Rev 200: 233-248.
Smith, A. L., J. N. F. Scott, and J. Boyes. 2019. The ESC: The Dangerous By-Product of V(D)J Recombination. Front Immunol 10: 1572.
Bories, J. C., J. M. Cayuela, P. Loiseau, and F. Sigaux. 1991. Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression. Blood 78: 2053- 2061.
Li, Y., P. M. Brauer, J. Singh, S. Xhiku, K. Yoganathan, J. C. Zuniga-Pflucker, and M. K. Anderson. 2017. Targeted Disruption of TCF12 Reveals HEB as Essential in Human Mesodermal Specification and Hematopoiesis. Stem Cell Reports 9: 779-795.
Kennedy, M., G. Awong, C. M. Sturgeon, A. Ditadi, R. LaMotte-Mohs, J. C. Zuniga- Pflucker, and G. Keller. 2012. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep 2: 1722-1735.
Villey, I., P. Quartier, F. Selz, and J. P. de Villartay. 1997. Germ-line transcription and methylation status of the TCR-J alpha locus in its accessible configuration. Eur J Immunol 27: 1619-1625.
Casero, D., S. Sandoval, C. S. Seet, J. Scholes, Y. Zhu, V. L. Ha, A. Luong, C. Parekh, and G. M. Crooks. 2015. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages. Nat Immunol 16: 1282-1291.
He, Y. W. 2000. The role of orphan nuclear receptor in thymocyte differentiation and lymphoid organ development. Immunol Res 22: 71-82.
Lefebvre, J. M., M. C. Haks, M. O. Carleton, M. Rhodes, G. Sinnathamby, M. C. Simon, L. C. Eisenlohr, L. A. Garrett-Sinha, and D. L. Wiest. 2005. Enforced expression of Spi- B reverses T lineage commitment and blocks beta-selection. J Immunol 174: 6184- 6194.
Klein, F., M. Mitrovic, J. Roux, C. Engdahl, L. von Muenchow, L. Alberti-Servera, H. J. Fehling, P. Pelczar, A. Rolink, and P. Tsapogas. 2019. The transcription factor Duxbl mediates elimination of pre-T cells that fail beta-selection. J Exp Med 216: 638-655.
Sicinska, E., I. Aifantis, L. Le Cam, W. Swat, C. Borowski, Q. Yu, A. A. Ferrando, S. D. Levin, Y. Geng, H. von Boehmer, and P. Sicinski. 2003. Requirement for cyclin D3 in lymphocyte development and T cell leukemias. Cancer Cell 4: 451-461. von Boehmer, H., I. Aifantis, J. Feinberg, O. Lechner, C. Saint-Ruf, U. Walter, J. Buer, and O. Azogui. 1999. Pleiotropic changes controlled by the pre-T-cell receptor. Curr Opin Immunol 11 : 135-142.
Michie, A. M., and J. C. Zuniga-Pflucker. 2002. Regulation of thymocyte differentiation: pre-TCR signals and beta-selection. Semin Immunol 14: 311-323.
Rothenberg, E. V., and T. Taghon. 2005. Molecular genetics of T cell development. Annu Rev Immunol 23: 601-649. Carrasco, Y. R., M. N. Navarro, V. G. de Yebenes, A. R. Ramiro, and M. L. Toribio. 2002. Regulation of surface expression of the human pre-T cell receptor complex. Semin Immunol 14: 325-334.
Blom, B., M. C. Verschuren, M. H. Heemskerk, A. Q. Bakker, E. J. van Gastel-Mol, I. L. Wolvers-Tettero, J. J. van Dongen, and H. Spits. 1999. TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation. Blood 93: 3033-3043.
Carrasco, Y. R., C. Trigueros, A. R. Ramiro, V. G. de Yebenes, and M. L. Toribio. 1999. Beta-selection is associated with the onset of CD8beta chain expression on
CD4(+)CD8alphaalpha(+) pre-T cells during human intrathymic development. Blood 94: 3491-3498.
Miyazaki, M., K. Miyazaki, M. Itoi, Y. Katoh, Y. Guo, R. Kanno, Y. Katoh-Fukui, H. Honda, T. Amagai, M. van Lohuizen, H. Kawamoto, and M. Kanno. 2008. Thymocyte proliferation induced by pre-T cell receptor signaling is maintained through polycomb gene product Bmi-1-mediated Cdkn2a repression. Immunity 28: 231-245.
Roszkowski, J. J., D. C. Yu, M. P. Rubinstein, M. D. McKee, D. J. Cole, and M. I. Nishimura. 2003. CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell. J Immunol 170: 2582-2589.
Benveniste, P. M., S. Roy, M. Nakatsugawa, E. L. Y. Chen, L. Nguyen, D. G. Millar, P. S. Ohashi, N. Hirano, E. J. Adams, and J. C. Zuniga-Pflucker. 2018. Generation and molecular recognition of melanoma-associated antigen-specific human gammadelta T cells. Sci Immunol 3.
Claims
1. A method of generating stem or progenitor cells unable to undergo T cell receptor (TCR) gene rearrangements (TCR), the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells.
2. The method of claim 1 , wherein the method further comprises (b) isolating cells of the T cell lineage.
3. The method of claim 1 or 2, wherein the at least one gene or protein required for
V(D)J recombination is RAG1 and/or RAG2.
4. The method of claim 1 or 2, wherein the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
5. The method of any one of claims 1 -4, wherein the stem cells are pluripotent stem cells.
6. The method of claim 5, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPSCs).
7. The method of any one of claims 1-6, wherein the stem cells or progenitor cells are human cells.
8. The method of any one of claims 1-7, wherein the cells of the T cell lineage are progenitor T (proT) cells.
9. The method of any one of claims 1-7, wherein the cells of the T cell lineage are
CD4+CD8+ double positive cells or CD4+CD8+CD3+ double positive cells.
10. The method of any one of claims 1-7, wherein the cells of the T cell lineage are CD8+CD3+ single positive cells or CD4+CD3+ single positive cells.
11. The method of any one of claims 1-10, further comprising engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise at least one of a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
12. The method of claim 11 , wherein the stem cells or progenitor cells or the cells of the T cell lineage express the at least one of the T cell receptor (TCR), the TCRp chain and the chimeric antigen receptor (CAR).
13. The method of any one of claims 1-10, further comprising engineering the stem cells or progenitor cells or the cells of the T cell lineage to comprise a nucleic acid encoding a TCRp chain.
14. The method of claim 13, wherein the stem cells or progenitor cells or the cells of the T cell lineage further comprise a nucleic acid encoding a CAR.
15. The method of claim 11 or 12, wherein the TCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
16. A cell of the T cell lineage, wherein the cell is generated by the method of any one of claims 1-15.
17. The cell of claim 16, wherein the cell of the T cell lineage is a CD4+CD8+ double positive cell or a CD4+CD8+CD3+ double positive cell.
18. The cell of claim 16, wherein the cell is a CD45+CD34+CD7+ progenitor T cell, CD8+CD3+ single positive cell or CD4+CD3+ single positive cell.
19. A stem or progenitor cell, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cell or progenitor cell is reduced or eliminated compared to a wild-type stem cell or progenitor cell.
20. The stem or progenitor cell of claim 19, wherein the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
21. The stem or progenitor cell of claim 20, wherein the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA- dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4
(XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
22. The stem or progenitor cell of any one of claims 19-21 , further comprising at least one of a nucleic acid encoding a T cell receptor (TCR), a TCRp chain and a chimeric antigen receptor (CAR).
23. The stem or progenitor cell of any one of claims 19-21 , further comprising a nucleic acid encoding a TCRp chain.
24. The stem or progenitor cell of any one of claims 19-23, wherein the stem cell is a pluripotent stem cell.
25. The stem or progenitor cell of claim 24, wherein the pluripotent stem cell is an embryonic stem cell or induced pluripotent stem cell (iPSC).
26. The stem or progenitor cell of any one of claims 19-25, wherein the stem cell or progenitor cell is a human cell.
27. A use of the stem or progenitor cell of any one of claims 19-26 for generating cells of the T cell lineage.
28. A kit comprising (i) a stem or progenitor cell of any one of claims 19-27 and (ii) instructions for use of the stem or progenitor cell of any one of claims 19-27 for generating cells of the T cell lineage.
29. A method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells or progenitor cells to a subject in need thereof, wherein the stem cells or progenitor cells are engineered to comprise at least one of a nucleic acid encoding a T cell receptor (TCR) and a chimeric antigen receptor (CAR) that confers specificity to an antigen.
30. A method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the T cell lineage wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the T cell lineage to a subject in need thereof, wherein the stem cells or progenitor cells or the cells of the T cell lineage are engineered to comprise at least one of a nucleic acid encoding a T cell receptor (TCR) and a chimeric antigen receptor (CAR) that confers specificity to an antigen.
31. The method of claim 29 or 30, wherein the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
32. The method of claim 29 or 30, wherein the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA- dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
33. The method of any one of claims 29-32, wherein the disease is cancer and the antigen is a tumor-associated antigen.
34. A method of generating stem or progenitor cells unable to undergo T cell receptor (BCR) gene rearrangements, the method comprising:
(a) culturing a sample comprising stem cells or progenitor cells, wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells.
35. The method of claim 34, wherein the method further comprises (b) isolating cells of the B cell lineage.
36. The method of claim 34 or 35, wherein the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
37. The method of claim 34 or 35, wherein the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA- dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
38. The method of any one of claims 34-37, wherein the stem cells are pluripotent stem cells.
39. The method of claim 38, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells (iPSCs).
40. The method of any one of claims 34-39, wherein the stem cells or progenitor cells are human cells.
41. The method of any one of claims 34-40, wherein the cells of the B cell lineage are CD20+ or CD19+ cells.
42. The method of any one of claims 34-40, wherein the cells of the B cell lineage are Tumor-Infiltrating B Cells (TIBs).
43. The method of any one of claims 34-42, further comprising engineering the stem cells or progenitor cells or the cells of the B cell lineage to comprise at least one of a nucleic acid encoding a B cell receptor (BCR), chimeric antigen receptor (CAR) or BCRp chain.
44. The method of claim 43, wherein the stem cells or progenitor cells or the cells of the B cell lineage express the B cell receptor (BCR), chimeric antigen receptor (CAR) or BCRp chain.
45. The method of claim 43 or 44, wherein the BCR or CAR confers specificity to an antigen, optionally a tumor-associated antigen, viral antigen or self antigen.
46. A cell of the B cell lineage, wherein the cell is generated by the method of any one of claims 35-45.
47. The cell of claim 46, wherein the cell of the B cell lineage is a CD20+ or CD19+ cell.
48. The cell of claim 46, wherein the cell of the B cell lineage is a Tumor-Infiltrating B Cell (TIB).
49. A use of the stem or progenitor cell of any one of claims 19-21 for generating cells of the B cell lineage.
50. A kit comprising (i) a stem or progenitor cell of any one of claims 19-21 and (ii) instructions for use of the stem or progenitor cell of any one of claims 19-21 for generating cells of the B cell lineage.
51. A method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the stem cells or progenitor cells to a subject in need thereof, wherein the stem cells or progenitor cells are engineered to comprise least one nucleic acid encoding a B cell receptor (BCR) or chimeric antigen receptor (CAR) that confers specificity to an antigen.
52. A method of treating a disease or condition in a subject comprising:
(i) culturing a sample comprising stem cells or progenitor cells, and isolating cells of the B cell lineage wherein expression of at least one gene or protein required for V(D)J recombination in the stem cells or progenitor cells is reduced or eliminated compared to wildtype stem cells or progenitor cells, and
(ii) administering an effective amount of the cells of the B cell lineage to a subject in need thereof,
wherein the stem cells or progenitor cells orthe cells of the B cell lineage are engineered to comprise least one nucleic acid encoding a B cell receptor (BCR) or chimeric antigen receptor (CAR) that confers specificity to an antigen.
53. The method of claim 51 or 52, wherein the at least one gene or protein required for V(D)J recombination is RAG1 and/or RAG2.
54. The method of claim 51 or 52, wherein the at least one gene or protein required for V(D)J recombination is selected from the group consisting of Artemis, DNA- dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1), Paralog of XRCC4 and XLF (PAXX), DNA polymerase l and DNA polymerase m.
55. The method of any one of claims 51-54, wherein the disease is cancer and the antigen is a tumor-associated antigen.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163178990P | 2021-04-23 | 2021-04-23 | |
PCT/CA2022/050622 WO2022221962A1 (en) | 2021-04-23 | 2022-04-22 | Stem cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4326856A1 true EP4326856A1 (en) | 2024-02-28 |
Family
ID=83723544
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP22790648.4A Pending EP4326856A1 (en) | 2021-04-23 | 2022-04-22 | Stem cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereof |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP4326856A1 (en) |
JP (1) | JP2024515697A (en) |
CN (1) | CN117480249A (en) |
CA (1) | CA3216417A1 (en) |
WO (1) | WO2022221962A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021514185A (en) * | 2018-02-14 | 2021-06-10 | サニーブルック リサーチ インスティチュート | Methods for generating cells of the T cell lineage |
-
2022
- 2022-04-22 JP JP2023564480A patent/JP2024515697A/en active Pending
- 2022-04-22 CN CN202280042484.9A patent/CN117480249A/en active Pending
- 2022-04-22 WO PCT/CA2022/050622 patent/WO2022221962A1/en active Application Filing
- 2022-04-22 CA CA3216417A patent/CA3216417A1/en active Pending
- 2022-04-22 EP EP22790648.4A patent/EP4326856A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
CA3216417A1 (en) | 2022-10-27 |
CN117480249A (en) | 2024-01-30 |
WO2022221962A1 (en) | 2022-10-27 |
JP2024515697A (en) | 2024-04-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108368520B (en) | Genome engineering of pluripotent cells | |
EP3612557B1 (en) | Antigen-specific immune effector cells | |
KR102598351B1 (en) | Method for direct differentiation of pluripotent stem cells into HLA homozygous immune cells | |
EP3134515B1 (en) | Application of induced pluripotent stem cells to generate adoptive cell therapy products | |
KR20180063333A (en) | Specified differentiation of pluripotent stem cells into immune cells | |
CN112512536A (en) | Ready-to-use cell therapy based on stem cell engineered INKT cells | |
CN113906133A (en) | CD3 reconstitution in engineered iPSCs and immune effector cells | |
CN114901693A (en) | Enhanced chimeric antigen receptors for immune effector cell engineering and uses thereof | |
US20200270581A1 (en) | Cellular reprogramming using temporal and transient plasmid vector expression system | |
CA3151781A1 (en) | Enhanced chimeric antigen receptor for immune effector cell engineering and use thereof | |
JP2023507118A (en) | Engineered cells for therapy | |
EP4326856A1 (en) | Stem cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereof | |
US20240209314A1 (en) | T cells comprising an unrearranged t cell receptor (tcr) gene locus and methods of use thereof | |
US20080112933A1 (en) | Methods and Compositions for Increasing Stem Cell Homing Using Gas Activators | |
WO2024077159A1 (en) | B cell lineages derived from pluripotent cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20231122 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |